Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5)

ABSTRACT

The invention provides methods of modifying feeding behavior, including increasing or decreasing food consumption, e.g., in connection with treating obesity, bulimia or anorexia. These methods involve administration of compounds that are selective agonists or antagonists for the Y5 receptor. One such compound has structure (I). In addition, this invention provides an isolated nucleic acid molecule encoding a Y5 receptor, an isolated Y5 receptor protein, vectors comprising an isolated nucleic acid molecule encoding a Y5 receptor, cells comprising such vectors, antibodies directed to the Y5 receptor, nucleic acid probes useful for detecting nucleic acid encoding Y5 receptors, antisense oligonucleotides complementary to any unique sequences of a nucleic acid molecule which encodes a Y5 receptor, and nonhuman transgenic animals which express DNA encoding a normal or a mutant Y5 receptor.

This application is a §317 of PCT International application No.PCT/US97/09504, filed Jun. 4, 1997, designating the United States ofAmerica, the contents of which are incorporated in its entirety into thepresent application.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citations for these publications may be found at the endof the specification, preceding the sequence listing and the claims.

Neuropeptide Y (NPY) is a member of the pancreatic polypeptide familywith widespread distribution throughout the mammalian nervous system.NPY and its relatives (peptide YY or PYY, and pancreatic polypeptide orPP) elicit a broad range of physiological effects through activation ofat least five G protein-coupled receptor subtypes known as Y1, Y2, Y3,Y4 (or PP), and the “atypical Y1”. The role of NPY as the most powerfulstimulant of feeding behavior yet described is thought to occurprimarily through activation of the hypothalamic “atypical Y1” receptor.This receptor is unique in that its classification was based solely onfeeding behavior data, rather than radioligand binding data, unlike theY1, Y2, Y3, and Y4 (or PP) receptors, each of which were describedpreviously in both radioligand binding and functional assays.

The peptide neurotransmitter neuropeptide Y (NPY) is a 36 amino acidmember of the pancreatic polypeptide family with widespread distributionthroughout the mammalian nervous system. NPY is considered to be themost powerful stimulant of feeding behavior yet described (Clark et al.,1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984). Directinjection into the hypothalamus of satiated rats, for example, canincrease food intake up to 10-fold over a 4-hour period (Stanley et al.,1992). The role of NPY in normal and abnormal eating behavior, and theability to interfere with NPY-dependent pathways as a means to appetiteand weight control, are areas of great interest in pharmacological andpharmaceutical research (Sahu and Kalra, 1993; Dryden et al., 1994). Anycredible means of studying or controlling NPY-dependent feedingbehavior, however, must necessarily be highly specific as NPY can actthrough at least 5 pharmacologically defined receptor subtypes to elicita wide variety of physiological functions (Dumont et al., 1992). It istherefore vital that knowledge of the molecular biology and structuraldiversity of the individual receptor subtypes be understood as part of arational drug design approach to develop subtype selective compounds. Abrief review of NPY receptor pharmacology is summarized below and alsoin Table 1.

TABLE 1 Pharmacologically Defined Receptors for NPY and RelatedPancreatic Polypeptides

Rank orders of affinity for key peptides (NPY, PYY, PP, [Leu³¹,Pro³⁴]NPY, NPY₂₋₃₆, and NPY₁₃₋₃₆) are based on previously reportedbinding and functional data (Schwartz et al., 1990; Wahlestedt et al.,1991; Dumont et al., 1992; Wahlestedt and Reis, 1993). Data for the Y2receptor were disclosed in PCT International Application No.PCT/US95/01469, filed Feb. 3, 1995, International Publication No. WO95/21245, published Aug. 10, 1995 the foregoing contents of which arehereby incorporated by reference. Data for the Y4 receptor weredisclosed in PCT International Application No. PCT/US94/14436 filed Dec.28, 1994, International Publication No. WO 95/17906, published Aug. 10,1995 the contents of which are hereby incorporated by reference. Missingpeptides in the series reflect a lack of published information. Table 1reflects current information obtained with cloned human Y1, Y2, Y4, andY5 receptors.

TABLE 1 Affinity (pK₁ or pEC₅₀) Receptor 11 to 10 10 to 9 9 to 8 8 to 77 to 6 <6 Y1 NPY NPY₂₋₃₆ NPY₁₃₋₃₆ PP PYY [Leu³¹, Pro³⁴]NPY Y2 PYYNPY₁₃₋₃₆ [Leu³¹, Pro³⁴]NPY NPY PP NPY₂₋₃₆ Y3 NPY [Pro³⁴]NPY NPY₁₃₋₃₆ PYYPP Y4 PP PYY NPY₂₋₃₆ NPY₁₃₋₃₆ [Leu³¹, Pro³⁴]NPY NPY Y5 or atypical PYYNPY₁₃₋₃₆ Y1 (feeding) NPY D-Trp³²NPY NPY₂₋₃₆ [Leu³¹, Pro³⁴]NPY

NPY Receptor Pharmacology

NPY receptor pharmacology has historically been based onstructure/activity relationships within the pancreatic polypeptidefamily. The entire family includes the namesake pancreatic polypeptide(PP), synthesized primarily by endocrine cells in the pancreas; peptideYY (PYY), synthesized primarily by endocrine cells in the gut; and NPY,synthesized primarily in neurons (Michel, 1991; Dumont et al., 1992;Wahlestedt and Reis, 1993). All pancreatic polypeptide family membersshare a compact structure involving a “PP-fold” and a conservedC-terminal hexapeptide ending in Tyr³⁶ (or Y³⁶ in the single lettercode). The striking conservation of Y³⁶ has prompted the reference tothe pancreatic polypeptides' receptors as “Y-type” receptors (Wahlestedtet al., 1987), all of which are proposed to function as seventransmembrane-spanning G protein-coupled receptors (Dumont et al.,1992).

The Y1 receptor recognizes NPY≧PYY>>PP (Grundemar et al., 1992). Thereceptor requires both the N- and the C-terminal regions of the peptidesfor optimal recognition. Exchange of Gln³⁴ in NPY or PYY with theanalogous residue from PP (Pro³⁴), however, is well-tolerated. The Y1receptor has been cloned from a variety of species including human, ratand mouse (Larhammar et al, 1992; Herzog et al, 1992; Eva et al, 1990;Eva et al, 1992). The Y2 receptor recognizes PYY˜NPY>>PP and isrelatively tolerant of N-terminal deletion (Grundemar et al., 1992). Thereceptor has a strict requirement for structure in the C-terminus(Arg³³-Gln³⁴-Arg³⁵-Tyr³⁶-NH₂); exchange of Gln³⁴ with Pro³⁴, as in PP,is not well tolerated. The Y2 receptor has recently been cloned. The Y3receptor is characterized by a strong preference for NPY over PYY and PP(Wahlestedt et al., 1991). [Pro³⁴]NPY is reasonably well tolerated eventhough PP, which also contains Pro³⁴, does not bind well to the Y3receptor. The Y3 receptor (Y3) has not yet been cloned. The Y4 receptorbinds PP>PYY>NPY. Like the Y1, the Y4 requires both the N- and theC-terminal regions of the peptides for optimal recognition. The“atypical Y1” or “feeding” receptor was defined exclusively by injectionof several pancreatic polypeptide analogs into the paraventricularnucleus of the rat hypothalamus which stimulated feeding behavior withthe following rank order: NPY₂₋₃₆≧NPY˜PYY˜[Leu³¹, Pro³⁴)NPY>NPY₁₃₋₃₆(Kalra et al., 1991; Stanley et al., 1992). The profile is similar tothat of a Y1-like receptor except for the anomalous ability of NPY₂₋₃₆to stimulate food intake with potency equivalent or better than that ofNPY. A subsequent report in J. Med. Chem. by Balasubramaniam et al.(1994) showed that feeding can be regulated by [D-Trp³²]NPY. While thispeptide was presented as an NPY antagonist, the published data at leastin part support a stimulatory effect of [D-Trp³²]NPY on feeding.[D-Trp³²]NPY thereby represents another diagnostic tool for receptoridentification. In contrast to other NPY receptor subtypes, the“feeding” receptor has never been characterized for peptide bindingaffinity in radioligand binding assays and the fact that a singlereceptor could be responsible for the feeding response has beenimpossible to validate in the absence of an isolated receptor protein;the possibility exists, for example, that the feeding response could bea composite profile of Y1 and Y2 subtypes.

This invention now reports the isolation by expression cloning of anovel Y-type receptor from a rat hypothalamic cDNA library, along withits pharmacological characterization, in situ localization, and humanhomolog. The data provided link this newly-cloned receptor subtype, fromnow on referred to as the Y5 subtype, to the “atypical Y1” feedingresponse. This discovery therefore provides a novel approach, throughthe use of heterologous expression systems, to develop a subtypeselective antagonist for obesity and other indications.

This invention is based on the use of a ¹²⁵I-PYY-based expressioncloning technique to isolate a rat hypothalamic cDNA encoding an“atypical Y1” receptor referred to herein as the Y5 receptor subtype.This application also concerns the isolation and characterization of aY5 homolog from human hippocampus.

Protein sequence analysis reveals that the Y5 receptor belongs to the Gprotein- coupled receptor superfamily. Both the human and rat homologdisplay≦42% identity in transmembrane domains with the previously cloned“Y-type” receptors. Rat brain localization studies using in situhybridization techniques verified the existence of Y5 receptor mRNA inrat hypothalamus. Pharmacological evaluation revealed the followingsimilarities between the Y5 and the “atypical Y1” receptor. 1) Peptidesbound to the Y5 receptor with a rank order of potency identical to thatdescribed for the feeding response: NPY≧NPY₂₋₃₆=PYY=[Leu³¹,Pro⁴]NPY>>NPY₁₃₋₃₆. 2) The Y5 receptor was negatively coupled to cAMPaccumulation, as had been proposed for the “atypical Y1” receptor. 3)Peptides activated the Y5 receptor with a rank order of potencyidentical to that described for the feeding response. 4) The reportedfeeding “modulator” [D-Trp³²]NPY bound selectively to the Y5 receptorand subsequently activated the receptor. 5) Both the Y5 and the“atypical Y1” receptors were sensitive to deletions or modifications inthe midregion of NPY and related peptide ligands. These data support theidentity of the Y5 receptor as the previously described “atypical Y1”,and furthermore indicate a role for the Y5 receptor as a potentialtarget in the treatment of obesity, metabolism, and appetite disorders.

The treatment of disorders or diseases associated with the inhibition ofthe Y5 receptor subtype, especially diseases caused by eating disorderslike obesity, bulimia nervosa, diabetes, dislipidimia, may be effectedby administration of compounds which bind selectively to the Y5 receptorand inhibit the activation of the Y5 receptor. Furthermore, any diseasestates in which the Y5 receptor subtype is involved, for example, memoryloss, epileptic seizures, migraine, sleep disturbance, pain, andaffective disorders such as depression and anxiety may also be treatedusing compounds which bind selectively to the Y5 receptor.

SUMMARY OF THE INVENTION

This invention provides a method of modifying a subject's feedingbehavior which comprises administering to the subject a compound whichis a Y5 receptor agonist or antagonist in an amount effective to alterthe subject's consumption of food and thereby modify the subject'sfeeding behavior.

This invention also provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non- peptidylcompound which is a Y5 receptor antagonist in an amount effective toinhibit the activity of the subject's Y5 receptor, wherein the bindingof the compound to the human receptor is characterized by a K_(i) lessthan 100 nanomolar when measured in the presence of ¹²⁵I-PYY in apredetermined amount.

Additionally, this invention provides a method of treating a subject'sfeeding disorder which comprises administering to the subject a peptidylcompound which is a Y5 receptor antagonist in an amount effective toinhibit the activity of the subject's Y5 receptor, wherein thecompound's binding to the human Y5 receptor is characterized by a K_(i)less than 10 nanomolar when measured in the presence of ¹²⁵I-PYY in apredetermined amount.

This invention further provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non-peptidylcompound which is a Y5 receptor agonist in an amount effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 100 nanomolar when measured in the presence of ¹²⁵I-PYYin a predetermined amount; a nd (b) the binding of the compound to anyother human Y-type receptor is characterized by a K_(i) greater than1000 nanomolar when measured in the presence of ¹²⁵I-PYY in apredetermined amount.

This invention also provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non-peptidylcompound which is a Y5 receptor agonist in an amount effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 1 nanomolar when measured in the presence of ¹²⁵I-PYY ina predetermined amount; and (b) the compound's binding to any otherhuman Y-type receptor is characterized by a K_(i) greater than 100nanomolar when measured in the presence of ¹²⁵I-PYY in a predeterminedamount.

This invention further provides a method of treating a subject's feedingdisorder which comprises administering to the subject a peptidylcompound which is a Y5 receptor agonist effective to increase theactivity of the subject's Y5 receptor, wherein (a) the binding of thecompound to the human Y5 receptor is characterized by a K_(i) less than1 nanomolar when measured in the presence of ¹²⁵I-PYY in a predeterminedamount; and (b) the binding of the compound to any other human Y-typereceptor is characterized by a K_(i) greater than 25 nanomolar whenmeasured in the presence of ¹²⁵I-PYY in a predetermined amount.

This invention provides a method of treating a subject's feedingdisorder which comprises administering to the subject a peptidylcompound which is a Y5 receptor agonist in an amount effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 0.1 nanomolar when measured in the presence of ¹²⁵I-PYYin a predetermined amount; and (b) the binding of the compound to anyother human Y-type receptor is characterized by a K_(i) greater than 1nanomolar when measured in the presence of ¹²⁵I-PYY in a predeterminedamount.

This invention further provides a method of treating a subject's feedingdisorder which comprises administering to the subject a peptidylcompound which is a Y5 receptor agonist in an amount effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 0.01 nanomolar when measured in the presence of ¹²⁵I-PYYin a predetermined amount; and (b) the binding of the compound to anyother human Y-type receptor is characterized by a K_(i) greater than 1nanomolar when measured in the presence of ¹²⁵I-PYY in a predeterminedamount.

Additionally, this invention provides an isolated nucleic acid encodinga Y5 receptor. This invention also provides an isolated Y5 receptorprotein. This invention provides a vector comprising the above-describednucleic acid.

This invention also provides a plasmid which comprises the regulatoryelements necessary for expression of DNA in a mammalian cell operativelylinked to the DNA encoding the human Y5 receptor as to permit expressionthereof designated pcEXV-hY5 (ATCC Accession No. 75943). This inventionfurther provides a plasmid which comprises the regulatory elementsnecessary for expression of DNA in a mammalian cell operatively linkedto the DNA encoding the rat Y5 receptor as to permit expression thereofdesignated pcEXV-rY5 (ATCC Accession No. 75944).

This invention provides a mammalian cell comprising the above-describedplasmid or vector.

This invention also provides a nucleic acid probe comprising a nucleicacid of at least 15 nucleotides capable of specifically hybridizing witha unique sequence included within the sequence of a nucleic acidencoding a Y5 receptor.

Additionally, this invention provides an antisense oligonucleotidehaving a sequence capable of specifically hybridizing to mRNA encoding aY5 receptor so as to prevent translation of the mRNA.

This invention also provides an antibody directed to a Y5 receptor.

This invention provides a pharmaceutical composition comprising anamount of the oligonucleotide effective to reduce activity of a human Y5receptor by passing through a cell membrane and binding specificallywith mRNA encoding a human Y5receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable carrier capable of passingthrough a cell membrane.

This invention also provides a pharmaceutical composition comprising anamount of an antagonist effective to reduce the activity of a human Y5receptor and a pharmaceutically acceptable carrier. This inventionfurther provides a pharmaceutical composition comprising an amount of anagonist effective to increase activity of a Y5 receptor and apharmaceutically acceptable carrier. This invention further provides theabove-described pharmaceutical composition which comprises an amount ofan antibody effective to block binding of a ligand to the Y5 receptorand a pharmaceutically acceptable carrier.

This invention additionally provides a transgenic nonhuman mammalexpressing DNA encoding a human Y5 receptor.

This invention also provides a method for determining whether a ligandcan specifically bind to a Y5 receptor which comprises contacting aplurality of cells transfected with and expressing DNA encoding the Y5receptor, or a membrane fraction from a cell extract of such cells, withthe ligand under conditions permitting binding of ligands to suchreceptor, detecting the presence of any such ligand specifically boundto the Y5 receptor, and thereby determining whether the ligandspecifically binds to the Y5 receptor.

This invention further provides a method for determining whether aligand is a Y5 receptor agonist which comprises contacting a celltransfected with and expressing nucleic acid encoding a human Y5receptor with the ligand under conditions permitting activation of theY5 receptor, detecting an increase in Y5 receptor activity, and therebydetermining whether the ligand is a human Y5 receptor agonist.

This invention provides a method for determining whether a ligand is aY5 receptor antagonist which comprises contacting a cell transfectedwith and expressing DNA encoding a Y5 receptor with the ligand in thepresence of a known Y5 receptor agonist, such as PYY or NPY, underconditions permitting the activation of the Y5 receptor, detecting adecrease in Y5 receptor activity, and thereby determining whether theligand is a Y5 receptor antagonist.

This invention further provides a method of screening a plurality ofchemical compounds not known to bind to a Y5 receptor to identify acompound which specifically binds to the Y5 receptor, which comprises(a) contacting a cell transfected with and expressing DNA encoding theY5 receptor, or a membrane fraction from a cell extract of such cells,with a compound known to bind specifically to the Y5 receptor; (b)contacting the preparation of step (a) with the plurality of compoundsnot known to bind specifically to the Y5 receptor, under conditionspermitting binding of compounds known to bind the Y5 receptor; (c)determining whether the binding of the compound known to bind to the Y5receptor is reduced in the presence of the compounds, relative to thebinding of the compound in the absence of the plurality of compounds;and if so (d) separately determining the binding to the Y5 receptor ofeach compound included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the Y5 receptor.

This invention also provides a method of screening a plurality ofchemical compounds not known to activate a Y5 receptor to identify acompound which activates the Y5 receptor which comprises (a) contactinga cell transfected with and expressing the Y5 receptor, or a membranefraction from a cell extract of such cells, with the plurality ofcompounds not known to bind specifically to the Y5 receptor, underconditions permitting activation of the Y5 receptor; (b) determiningwhether the activity of the Y5 receptor is increased in the presence ofthe compounds; and if so (c) separately determining whether theactivation of the Y5 receptor is increased by each compound included inthe plurality of compounds, so as to thereby identify the compound whichactivates the Y5 receptor.

This invention further provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a Y5 receptorto identify a compound which inhibits the activation of the Y5 receptor,which comprises (a) contacting a cell transfected with and expressingthe Y5 receptor, or a membrane fraction from a cell extract of suchcells, with the plurality of compounds in the presence of a known Y5receptor agonist, under conditions permitting activation of the Y5receptor; (b) determining whether the activation of the Y5 receptor isreduced in the presence of the plurality of compounds, relative to theactivation of the Y5 receptor in the absence of the plurality ofcompounds; and if so (c) separately determining the inhibition ofactivation of the Y5 receptor for each compound included in theplurality of compounds, so as to thereby identify the compound whichinhibits the activation of the Y5 receptor.

Additionally, this invention provides a process for identifying achemical compound which specifically binds to a Y5 receptor, whichcomprises contacting nonneuronal cells expressing on their cell surfacethe Y5 receptor, or a membrane fraction from a cell extract of suchcells, with the chemical compound under conditions suitable for binding,and detecting specific binding of the chemical compound to the Y5receptor.

This invention also provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a Y5receptor which comprises separately contacting nonneuronal cellsexpressing on their cell surface a Y5 receptor, or a membrane fractionfrom a cell extract of such cells, with both the chemical compound and asecond chemical compound known to bind to the receptor, and with onlythe second chemical compound, under conditions suitable for binding ofboth compounds, and detecting specific binding of the chemical compoundto the Y5 receptor, a decrease in the binding of the second chemicalcompound to the Y5 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the Y5 receptor.

This invention further provides a process for determining whether achemical compound specifically binds to and activates a Y5 receptor,which comprises contacting nonneuronal cells producing a secondmessenger response and expressing on their cell surface a Y5 receptor,or a membrane fraction from a cell extract of such cells, with thechemical compound under conditions suitable for activation of the Y5receptor, and measuring the second messenger response in the presenceand in the absence of the chemical compound, a change in secondmessenger response in the presence of the chemical compound indicatingthat the chemical compound activates the Y5 receptor.

This invention also provides a process for determining whether achemical compound specifically binds to and inhibits activation of a Y5receptor, which comprises separately contacting nonneuronal cellsproducing a second messenger response and expressing on their cellsurface a Y5 receptor, or a membrane fraction from a cell extract ofsuch cells, with both the chemical compound and a second chemicalcompound known to activate the Y5 receptor, and with only the secondchemical compound, under conditions suitable for activation of the Y5receptor, and measuring the second messenger response in the presence ofonly the second chemical compound and in the presence of both the secondchemical compound and the chemical compound, a smaller change in secondmessenger response in the presence of both the chemical compound and thesecond chemical compound indicating that the chemical compound inhibitsactivation of the Y5 receptor.

This invention additionally provides a method of treating a subject'sabnormality, wherein the abnormality is alleviated by the inhibition ofa Y5 receptor which comprises administering to a subject an effectiveamount of Y5 receptor antagonist. This invention also provides a methodof treating a subject's abnormality wherein the abnormality isalleviated by the activation of a Y5 receptor which comprisesadministering to a subject an effective amount of a Y5 receptor agonist.

This invention further provides a method for diagnosing a predispositionto a disorder associated with the activity of a specific allelic form ofa human Y5 receptor which comprises: a. obtaining DNA from a subject tobe tested; digesting the DNA with restriction enzymes; c. separating theresulting DNA fragments; d. contacting the fragments with a detectablylabeled nucleic acid probe capable of specifically hybridizing with asequence uniquely present within the sequence of a nucleic acid moleculeencoding the allelic form of the human Y5 receptor; and e. detecting thepresence of labeled probe from the subject to be tested, the presence ofsuch hybridized probe indicating that the subject is predisposed to thedisorder.

This invention also provides a method of preparing the isolated Y5receptor which comprises: a. inserting nucleic acid encoding Y5 receptorin a suitable vector which comprises the regulatory elements necessaryfor expression of the nucleic acid operatively linked to the nucleicacid encoding a Y5 receptor; b. inserting the resulting vector in asuitable host cell so as to obtain a cell which produces the Y5receptor; c. recovering the receptor produced by the resulting cell; andd. purifying the receptor so recovered.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Competitive displacement of ¹²⁵I-PYY on membranes from rathypothalamus. Membranes were incubated with ¹²⁵I-PYY and increasingconcentrations of peptide competitors. IC₅₀ values corresponding to 50%displacement were determined by nonlinear regression analysis. Data arerepresentative of at least two independent experiments. IC₅₀ values forthese compounds are listed separately in Table 2.

FIG. 2 Competitive displacement of ¹²⁵I-PYY₃₋₃₆ on membranes from rathypothalamus. Membranes were incubated with ¹²⁵I-PYY₃₋₃₆ and increasingconcentrations of peptide competitors. IC₅₀ values corresponding to 50%displacement were determined by nonlinear regression analysis. Data arerepresentative of at least two independent experiments. IC₅₀ values forthese compounds are listed separately in Table 2.

FIG. 3 Nucleotide sequence of the rat hypothalamic Y5 cDNA clone (Seq.I.D. No 1). Initiation and stop codons are underlined. Only partial 5′and 3′ untranslated sequences are shown.

FIG. 4 Corresponding amino acid sequence of the rat hypothalamic Y5 cDNAclone (Seq. I.D. No. 2).

FIG. 5 Nucleotide sequence of the human hippocampal Y5 cDNA clone (Seq.I.D. No. 3). Initiation and stop codons are underlined. Only partial 5′and 3′ untranslated sequences are shown.

FIG. 6 Corresponding amino acid sequence of the human hippocampal Y5cDNA clone(Seq. I.D. No. 4).

FIGS. 7A-E. Comparison of coding nucleotide sequences between rathypothalamic Y5 (top row) and human hippocampal Y5 (bottom row) cDNAclones (84.1% nucleotide identity). FIGS. F-G. Comparison of deducedamino acid sequences between rat hypothalamic Y5 (top row) and humanhippocampal Y5 (bottom row) cDNA clones (87.2% overall and 98.8%transmembrane domain identities).

FIGS. 8A-C Comparison of the human Y5 receptor deduced amino acidsequence with those of the human Y1, Y2, Y4 sequences. Solid bars, theseven putative membrane-spanning domains (TM I-VII). Shading, identitiesbetween receptor sequences.

FIG. 9 Equilibrium binding of ¹²⁵I-PYY to membranes from COS-7 cellstransiently expressing rat Y5 receptors. Membranes were incubated with¹²⁵I-PYY for the times indicated, in the presence or absence of 300 nMhuman NPY. Specific binding, B, was plotted against time, t, to obtainthe maximum number of equilibrium binding sites, B_(max), and observedassociation rate, K_(obs), according to the equation,B=B_(max)*(1−e^(−(kobs*t))). Binding is shown as the percentage of totalequilibrium binding, B_(max), determined by nonlinear regressionanalysis. Each point represents a triplicate determination.

FIG. 10 Saturable equilibrium binding of ¹²⁵I-PYY to membranes fromCOS-7 cells transiently expressing rat Y5 receptors. Membranes wereincubated with ¹²⁵I-PYY ranging in concentration from 0.4 pM to 2.7 nM,in the presence or absence of 300 nM human NPY. Specific binding, B, wasplotted against the free ¹²⁵I-PYY concentration, [L], to obtain themaximum number of saturable binding sites, B_(max), and the ¹²⁵I-PYYequilibrium dissociation constant, K_(d), according to the bindingisotherm, B=B_(max)[L]/([L]+K_(d)). Specific binding is shown. Data arerepresentative of three independent experiments, with each pointmeasured in triplicate.

FIG. 11 Competitive displacement of ¹²⁵I-PYY from COS-7 cellstransiently expressing rat Y5 receptors. Membranes were incubated with¹²⁵I-PYY and increasing concentrations of peptide competitors. IC₅₀values corresponding to 50% displacement were determined by nonlinearregression analysis and converted to K_(i) values according to theequation, K_(i)=IC₅₀/(1+[L]/K_(d)), where [L] is the ¹²⁵I-PYYconcentration and K_(d) is the equilibrium dissociation constant of¹²⁵I-PYY. Data are representative of at least two independentexperiments. Rank orders of affinity for these and other compounds arelisted separately in Table 4.

FIG. 12 Inhibition of forskolin-stimulated cAMP accumulation in intact293 cells stably expressing rat Y5 receptors. Functional data werederived from radioimmunoassay of cAMP in 293 cells stimulated with 10 μMforskolin over a 5 minute period. Rat/human NPY was tested for agonistactivity at concentrations ranging from 0.03 pM to 0.3 μM over the sameperiod. The EC₅₀ value corresponding to 50% maximal activity wasdetermined by nonlinear regression analysis. The data shown arerepresentative of three independent experiments.

FIGS. 13A-H Schematic diagrams of coronal sections through the ratbrain, illustrating the distribution of NPY Y5 receptor mRNA, asvisualized microscopically in sections dipped in liquid emulsion. Thesections are arranged from rostral (A) to caudal (H). Differences insilver grain density over individual neurons in a given area areindicated by the hatching gradient. The full definitions for theabbreviations are as follows:

Aco=anterior cortical amygdaloid nucleus;

AD=anterodorsal thalamic nucleus;

APT=anterior pretectal nucleus;

Arc=arcuate hypothalamic nucleus;

BLA=basolateral amygdaloid nucleus anterior;

CA3=field CA3 of Ammon's horn, hippocampus;

CeA=central amygdaloid nucleus;

Cg=cingulate cortex;

CL=centrolateral thalamic nucleus;

CM=central medial thalamic nucleus

DG=dentate gyrus, hippocampus;

DMH=dorsomedial hypothalamic nucleus;

DR=dorsal raphe;

GiA=gigantocellular reticular nucleus, alpha;

HDB=nucleus horizontal limb diagonal band;

InG=intermediate gray layer superior colliculus;

LC=locus coeruleus;

LH=lateral hypothalamic area;

MePV=medial amygdaloid nucleus, posteroventral;

MVe=medial vestibular nucleus;

MHb=medial habenular nucleus;

MPN=medial preoptic nucleus;

PAG=periaqueductal gray;

PaS=parasubiculum;

PC=paracentral thalamic nucleus;

PCRtA=parvocellular reticular nucleus, alpha;

Pe=periventricular hypothalamic nucleus;

PrS=presubiculum;

PN=pontine nuclei;

PVH=paraventricular hypothalamic nucleus;

PVHmp=paraventricular hypothalamic nucleus, medial parvicellular part

PVT=paraventricular thalamic nucleus;

Re=reunions thalamic nucleus;

RLi=rostral linear nucleus raphe;

RSG=retrosplenial cortex;

SCN=suprachiasmatic nucleus;

SNc=substantia nigra, pars compacta; and

SON=supraoptic nucleus.

FIG. 14 Partial Nucleotide sequence of the canine Y5 cDNA clonebeginning immediately upstream of TM III to the stop codon (underlined).(Seq. I.D. No 5). Only partial untranslated sequences are shown.

FIG. 15 Corresponding partial amino acid sequence of the canine Y5 cDNAclone (Seq. I.D. No. 6).

FIG. 16 A. Northern blot analysis of various rat tissues. FIG. B.Northern blot analysis of various human brain areas: amygdala, caudatenucleus, corpus callosum, hippocampus, whole brain, substantia nigra,subthalamic nucleus, and thalamus. FIG. C. Northern blot analysis ofvarious additional human brain areas: cerebellum, cerebral cortex,medulla, spinal cord, occipital lobe, frontal lobe, temporal lobe, andputamen. Hybridization was done under conditions of high stringency, asdescribed in Experimental Details.

FIGS. 17A-B Southern blot analysis of human(A) or rat(B) genomic DNAencoding the Y5 receptor subtype. Hybridization was done underconditions of high stringency, as described in Experimental Details.

FIG. 18 Time course for equilibrium binding of ¹²⁵I-Leu³¹, Pro³⁴-PYY tothe rat Y5 receptor. Membranes were incubated with 0.08 nM radioligandat room temperature for the length of time indicated in binding buffercontaining either 10 mM Na+ or 138 mM Na+.

FIG. 19 Guanine Nucleotide Modulation of Y5 Peptide Binding. Human orrat Y5 receptors transiently expressed in COS-7 cell membranes, or humanY5 receptors stably expressed in LM(tk-) cell membranes, were incubatedwith 0.08 nM ¹²⁵I-PYY and increasing concentrations of Gpp(NH)p asindicated under standard binding assay conditions. Radioligand bindingis reported as cpm, efficiency=0.8. For the human Y5 in LM(tk-) (0.007mg membrane protein/sample), the maximum Δ cpm=−2343. Given a specificactivity of 2200 Ci/mmol, the change in radioligand binding is thereforecalculated to be −0.6 fmol/0.007 mg protein=−85 fmol/mg membraneprotein.

FIG. 20 NPY-Dependent Inhibition of Forskolin Stimulated cAMPAccumulation by Cloned Y5 Receptors. Intact cells stably transfectedwith human or rat Y5 receptors were incubated with forskolin plus arange of human NPY concentrations as indicated. A representativeexperiment is shown for each receptor system (n≧2).

FIGS. 21A-D Calcium Mobilization: Fura-2 Assay. Cloned human Y-typereceptors in the host cells indicated were screened for intracellularcalcium mobilization in response to NPY and related peptides.Representative calcium transients are shown for each receptor system.

A. Human Y1 receptor

B. Human Y2 receptor

C. Human Y4 receptor

D. Human Y5 receptor

FIGS. 22A-C Structures of Y5-selective compounds. The structures of thecompounds evaluated at the human Y-type receptors are given.

FIG. 23 Nucleotide sequence of the canine Y5 cDNA clone (Seq. I.D. No.13). Initiation and stop codons are underlined. Only partial 5′ and 3′untranslated sequences are shown.

FIG. 24 Corresponding amino acid sequence of the canine Y5 cDNA clone(Seq. I.D. No. 14).

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the following standard abbreviations areused to indicate specific nucleotide bases:

C = cytosine A = adenine T = thymine G = guanine

Furthermore, the term “agonist” is used throughout this application toindicate any peptide or non-peptidyl compound which increases theactivity of any of the receptors of the subject invention. The term“antagonist” is used throughout this application to indicate any peptideor non-peptidyl compound which decreases or inhibits the activity of anyof the receptors of the subject invention.

The activity of a G-protein coupled receptor such as a Y5 receptor maybe measured using any of a variety of appropriate functional assays inwhich activation of the receptor in question results in an observablechange in the level of some second messenger system, including but notlimited to adenylate cyclase, calcium mobilization, inositolphospholipid hydrolysis or guanylyl cyclase.

This invention provides a method of modifying a subject's feedingbehavior which comprises administering to the subject a compound whichis a Y5 receptor agonist or antagonist in an amount effective to alterthe subject's consumption of food and thereby modify the subject'sfeeding behavior. In one embodiment, the compound is a Y5 receptorantagonist and the amount is effective to decrease the consumption offood by the subject. In a further embodiment, the compound isadministered in combination with food. In another embodiment thecompound is a Y5 receptor agonist and the amount is effective toincrease the consumption of food by the subject. In a further embodimentthe compound is administered in combination with food. The subject maybe a vertebrate, a mammal, a human or a canine subject.

This invention also provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non-peptidylcompound which is a Y5 receptor antagonist in an amount effective toinhibit the activity of the subject's Y5 receptor, wherein the bindingof the compound to the human Y5 receptor is characterized by a K_(i)less than 100 nanomolar when measured in the presence of ¹²⁵I-PYY at apredetermined concentration. In one embodiment the compound has a K_(i)less than 50 nanomolar. In another embodiment, the compound has a K_(i)less than 10 nanomolar. In a further embodiment, the binding of thecompound to any other human Y-type receptor is characterized by a K_(i)greater than 10 nanomolar when measured in the presence of ¹²⁵I-PYY at apredetermined concentration. In another embodiment, the binding of thecompound to any other human Y-type receptor is characterized by a K_(i)greater than 50 nanomolar. In another embodiment, the binding of thecompound is characterized by a K_(i) greater than 100 nanomolar. In oneembodiment, the compound binds to the human Y5 receptor with an affinitygreater than ten-fold higher than the affinity with which the compoundbinds to any other human Y-type receptor. In a further embodiment thecompound binds to the human Y5 receptor with an affinity greater thanten-fold higher than the affinity with which the compound binds to eachof the human Y1, human Y2 and human Y4 receptors. The feeding disordermay be obesity or bulimia. The subject may be a vertebrate, a mammal, ahuman or a canine subject.

This invention further provides a method of treating a subject's feedingdisorder which comprises administering to the subject a peptidylcompound which is a Y5 receptor antagonist in an amount effective toinhibit the activity of the subject's Y5 receptor, wherein thecompound's binding to the human Y5 receptor is characterized by a K_(i)less than 10 nanomolar when measured in the presence of ¹²⁵I-PYY at apredetermined concentration. In one embodiment, the compound's bindingis characterized by a K_(i) less than 1 nanomolar. In anotherembodiment, the compound's binding to any other human Y-type receptor ischaracterized by a K_(i) greater than 10 nanomolar when measured in thepresence of ¹²⁵I-PYY at a predetermined concentration. In anotherembodiment the compound's binding to each of the human Y1, human Y2, andhuman Y4 receptors is characterized by a K_(i) greater than 10 nanomolarwhen measured in the presence of ¹²⁵I-PYY at a predeterminedconcentration. In a further embodiment, the compound's binding to anyother human Y-type receptor is characterized by a K_(i) greater than 50nanomolar. In another embodiment the compound's binding to any otherhuman Y-type receptor is characterized by a K_(i) greater than 100nanomolar. In one embodiment, the compound binds to the human Y5receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to any other human Y-type receptor. Inanother embodiment, the compound binds to the human Y5 receptor with anaffinity greater than ten-fold higher than the affinity with which thecompound binds to each of the human Y1, human Y2, and human Y4receptors. The feeding disorder may be obesity or bulimia. The subjectmay be a vertebrate, a mammal, a human, or a canine subject.

This invention provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non-peptidylcompound which is a Y5 receptor agonist in an amount effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 100 nanomolar when measured in the presence of ¹²⁵I-PYYat a predetermined concentration; and (b) the binding of the compound toany other human Y-type receptor is characterized by a K_(i) greater than1000 nanomolar when measured in the presence of ¹²⁵I-PYY at apredetermined concentration. In one embodiment, the binding of thecompound to the human Y5 receptor is characterized by a K_(i) less than10 nanomolar.

This invention also provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non-peptidylcompound which is a Y5 receptor agonist in an amount effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 1 nanomolar when measured in the presence in ¹²⁵I-PYY;and (b) the compound's binding to any other human Y-type receptor ischaracterized by a K_(i) greater than 100 nanomolar when measured in thepresence of ¹²⁵I-PYY at a predetermined concentration. In oneembodiment, the compound binds to the human Y5 receptor with an affinitygreater than ten-fold higher than the affinity with which the compoundbinds to any other human Y-type receptor. In another embodiment, thecompound binds to the human Y5 receptor with an affinity greater thanten-fold higher than the affinity with which the compound binds to eachof the human Y1, human Y2, and human Y4 receptors. The feeding disordermay be anorexia. The subject may be a vertebrate, a mammal, a human, ora canine subject.

This invention further provides a method of treating a subject's feedingdisorder which comprises administering to the subject a peptidylcompound which is a Y5 receptor agonist in an amount effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 1 nanomolar when measured in the presence of ¹²⁵I-PYY ata predetermined concentration; and (b) the binding of the compound toany other human Y-type receptor is characterized by a K_(i) greater than25 nanomolar when measured in the presence of ¹²⁵I-PYY at apredetermined concentration.

This invention provides a method of treating a subject's feedingdisorder which comprises administering to the subject a peptidylcompound which is a Y5 receptor agonist in an amount effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 0.1 nanomolar when measured in the presence of ¹²⁵I-PYYat a predetermined concentration; and (b) the binding of the compound toany other human Y-type receptor is characterized by a K_(i) greater than1 nanomolar when measured in the presence of ¹²⁵I-PYY at a predeterminedconcentration. In one embodiment, the binding of the agonist to anyother human Y-type receptor is characterized by a K_(i) greater than 10nanomolar.

This invention provides a method of treating a subject's feedingdisorder which comprises administering to the subject a peptidylcompound which is a Y5 receptor agonist in an amount effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 0.01 nanomolar when measured in the presence of ¹²⁵I-PYYat a predetermined concentration; and (b) the binding of the compound toany other human Y-type receptor is characterized by a K_(i) greater than1 nanomolar when measured in the presence of ¹²⁵I-PYY at a predeterminedconcentration. In one embodiment, the compound binds to the human Y5receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to any other human Y-type receptor.

In another embodiment, the compound binds to the human Y5 receptor withan affinity greater than ten-fold higher than the affinity with whichthe compound binds to each of the human Y1, human Y2, and human Y4receptors. In one embodiment, the feeding disorder is anorexia. Thesubject may be a vertebrate, a mammal, a human, or a canine subject.

In addition, this invention provides an isolated nucleic acid encoding aY5 receptor. In one embodiment, the Y5 receptor is a vertebrate or amammalian Y5 receptor. In another embodiment, the Y5 receptor is a humanY5 receptor. In a further embodiment, the isolated nucleic acid encodesa receptor being characterized by an amino acid sequence in thetransmembrane region, wherein the amino acid sequence has 60% homologyor higher to the amino acid sequence in the transmembrane region of thehuman Y5 receptor shown in FIG. 6. In another embodiment, the Y5receptor has substantially the same amino acid sequence as described inFIG. 4. In another embodiment, the Y5 receptor has substantially thesame amino acid sequence as described in FIG. 6. In another embodiment,the Y5 receptor has substantially the same amino acid sequence asdescribed in FIG. 24.

This invention provides the above-described isolated nucleic acid,wherein the nucleic acid is a DNA. In an embodiment, the DNA is a cDNA.In another embodiment, the DNA is a genomic DNA. In still anotherembodiment, the nucleic acid is RNA. In a separate embodiment, thenucleic acid encodes a human Y5 receptor. In an embodiment, the human Y5receptor has the amino acid sequence as described in FIG. 6. In anotherembodiment, the nucleic acid encodes a rat Y5 receptor. In anembodiment, the rat Y5 receptor has the amino acid sequence as shown inFIG. 4. In another embodiment, the nucleic acid encodes a canine Y5receptor. In an embodiment, the canine Y5 receptor has the amino acidsequence shown in FIG. 24.

This invention further provides DNA which is degenerate with any of theDNA shown in FIGS. 3, 5, 14 and 23, wherein the DNA encodes Y5 receptorshaving the amino acid sequences shown in FIGS. 4, 6, 15 and 24,respectively.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of Y5 receptor, but which should notproduce phenotypic changes. Alternatively, this invention alsoencompasses DNAs and cDNAs which hybridize to the DNA, RNA, and cDNA ofthe subject invention. Hybridization methods are well known to those ofskill in the art.

The nucleic acid of the subject invention also includes nucleic acidcoding for polypeptide analogs, fragments or derivatives of antigenicpolypeptides which differ from naturally-occurring forms in terms of theidentity or location of one of more amino acid residues (deletionanalogs containing less than all of the residues specified for theprotein, substitution analogs wherein one or more residues specified arereplaced by other residues and addition analogs where in one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These nucleic acids include: theincorporation of codons “preferred” for expression by selectednon-mammalian hosts; the provision of sites for cleavage by restrictionendonuclease enzymes; and the provision of additional initial, terminalor intermediate nucleic acid sequences that facilitate construction ofreadily expressed vectors.

The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The nucleic acid isuseful for generating new cloning and expression vectors, transformedand transfected prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

In a separate embodiment, the nucleic acid encodes a rat Y5 receptor. Inanother embodiment, the rat Y5 receptor has the amino acid sequenceshown in FIG. 4.

This invention also provides a n isolated Y5 receptor protein. In oneembodiment, the Y5 receptor protein is a human Y5 receptor protein. Inanother embodiment, the human Y5 receptor protein has the amino acidsequence as shown in FIG. 6. In a further embodiment, the Y5 receptorprotein is a rat Y5 receptor protein. In another embodiment, the rat Y5receptor protein has the amino acid sequence as shown in FIG. 4. Inanother embodiment, the Y5 receptor protein is a canine Y5 receptorprotein. In a further embodiment, the canine Y5 receptor protein has theamino acid sequence as shown in FIG. 24.

This invention provides a vector comprising the above-described nucleicacid.

Vectors which comprise the isolated nucleic acid described hereinabovealso are provided. Suitable vectors comprise, but are not limited to, aplasmid or a virus. These vectors may be transform ed into a suitablehost cell to form a host cell vector system for the production of apolypeptide having the biological activity of a Y5 receptor.

This invention provides the above-described vector adapted forexpression in a cell which further comprises the regulatory elementsnecessary for expression of the nucleic acid in the cell operativelylinked to the nucleic acid encoding the Y5 receptor as to permitexpression thereof. In an embodiment, the cell is a Xenopus cell such asan oocyte or melanophore.

This invention provides the above-described vector adapted forexpression in a bacterial cell which further comprises the regulatoryelements necessary for expression of the nucleic acid in the bacterialcell operatively linked to the nucleic acid encoding the Y5 receptor asto permit expression thereof.

This invention provides the above-described vector adapted forexpression in a yeast cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the yeast celloperatively linked to the nucleic acid encoding the Y5 receptor as topermit expression thereof.

This invention provides the above-described vector adapted forexpression in an insect cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the insect celloperatively linked to the nucleic acid encoding the Y5 receptor as topermit expression thereof.

In an embodiment, the vector is adapted for expression in a mammaliancell which comprises the regulatory elements necessary for expression ofthe nucleic acid in the mammalian cell operatively linked to the nucleicacid encoding the mammalian Y5 receptor as to permit expression thereof.

In an embodiment, the vector is adapted for expression in a mammaliancell which comprises the regulatory elements necessary for expression ofthe nucleica acid in the mammalian cell operatively linked to thenucleic acid encoding the canine Y5 receptor as to permit expressionthereof.

In a further embodiment, the vector is adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the human Y5 receptor as to permitexpression thereof.

In a still further embodiment, the plasmid is adapted for expression ina mammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the rat Y5 receptor as to permit expressionthereof.

In a still further embodiment, the plasmid is adapted for expression ina mammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the canine Y5 receptor as to permitexpression thereof.

This invention provides the above-described plasmid adapted forexpression in a mammalian cell which comprises the regulatory elementsnecessary for expression of nucleic acid in a mammalian cell operativelylinked to the nucleic acid encoding the mammalian Y5 receptor as topermit expression thereof.

This invention provides a plasmid which comprises the regulatoryelements necessary for expression of nucleic acid in a mammalian celloperatively linked to the nucleic acid encoding the human Y5 receptor asto permit expression thereof designated pcEXV-hY5 (ATCC Accession No.75943).

This plasmid (pcEXV-hY5) was deposited on Nov. 4, 1994 with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and was accorded ATCC Accession No. 75943.

This invention provides a plasmid which comprises the regulatoryelements necessary for expression of nucleic acid in a mammalian celloperatively linked to the nucleic acid encoding the rat Y5 receptor asto permit expression thereof designated pcEXV-rY5 (ATCC Accession No.75944).

This plasmid (pcEXV-rY5) was deposited on Nov. 4, 1994 with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and was accorded ATCC Accession No. CRL75944.

This invention provides a plasmid designated Y5-bd-5 (ATCC Accession No.97355). This invention also provides a plasmid designated Y5-bd-8 (ATCCAccession No. 97354). These plasmids were deposited on Dec. 1, 1995 withthe American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This invention further provides aplasmid designated cY5-BO11, which comprises a canine Y5 receptor. Thisplasmid was deposited on May 29, 1996 with the ATCC under the provisionsof the Budapest Treaty for the International Recognition of the Depositof Microorganism for the Purposes of Patent procedure, and was accordedATCC Accession No. 97587.

This invention provides a baculovirus designated hY5-BB3 (ATCC AccessionNo. VR-2520). This baculovirus was deposited on Nov. 15, 1995 with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., 20852, U.S.A. under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and was accordedATCC Accession No. VR-2520.

This invention provides a mammalian cell comprising the above-describedplasmid or vector. In an embodiment, the mammalian cell is a COS-7 cell,a Chinese hamster ovary (CHO) cell, or a neuronal cell such as the glialcell line C6.

In another embodiment, the mammalian cell is a 293 human embryonickidney cell designated 293-rY5-14 (ATCC Accession No. CRL 11757). Thiscell (293-rY5-14) was deposited on Nov. 4, 1994 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure and was accorded ATCC Accession No. CRL 11757.

In a further embodiment, the mammalian cell is a mouse fibroblastLM(tk-) cell, containing the plasmid pcEXV-hY5 and designated L-hY5-7(ATCC Accession No. CRL-11995). In another embodiment, the mammaliancell is a mouse embryonic NIH-3T3 cell containing the plasmid pcEXV-hY5and designated N-hY5-8 (ATCC Accession No. CRL-11994). These cells weredeposited on Nov. 15, 1995 with the American Type Culture Collection(ATCC) 12301 Parklawn Drive, Rockville, Md., 20852, U.S.A. under theprovisions of the Budapest Treaty for the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure, andwere accorded ATCC Accession Nos. CRL-11995 and CRL-11994, respectively.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a unique sequence included within the sequence of a nucleic acidmolecule encoding a Y5 receptor. In an embodiment, the nucleic acid isDNA.

This nucleic acid produced can either be DNA or RNA. As used herein, thephrase “specifically hybridizing” means the ability of a nucleic acid torecognize a nucleic acid sequence complementary to its own and to formdouble-helical segments through hydrogen bonding between complementarybase pairs.

This nucleic acid of at least 15 nucleotides capable of specificallyhybridizing with a sequence of a nucleic acid encoding the human Y5receptors can be used as a probe. Nucleic acid probe technology is wellknown to those skilled in the art who will readily appreciate that suchprobes may vary greatly in length and may be labeled with a detectablelabel, such as a radioisotope or fluorescent dye, to facilitatedetection of the probe. DNA probe molecules may be produced by insertionof a DNA molecule which encodes the Y5 receptor into suitable vectors,such as plasmids or bacteriophages, followed by transforming intosuitable bacterial host cells, replication in the transformed bacterialhost cells and harvesting of the DNA probes, using methods well known inthe art. Alternatively, probes may be generated chemically from DNAsynthesizers.

RNA probes may be generated by inserting the DNA which encodes the Y5receptor downstream of a bacteriophage promoter such as T3, T7 or SP6.Large amounts of RNA probe may be produced by incubating the labelednucleotides with the linearized fragment where it contains an upstreampromoter in the presence of the appropriate RNA polymerase.

This invention also provides a nucleic acid of at least 15 nucleotidescapable of specifically hybridizing with a sequence of a nucleic acidwhich is complementary to the mammalian nucleic acid encoding a Y5receptor. This nucleic acid may either be a DNA or RNA molecule. Thisinvention further provides a nucleic acid probe molecule of at least 15nucleotides which is complementary to a unique fragment of the sequenceof the nucleic acid molecule encoding a Y5 receptor. This invention alsoprovides a nucleic acid probe comprising a nucleic acid molecule of atleast 15 nucleotides which is complementary to the antisense sequence ofa unique fragment of the sequence of a nucleic acid molecule encoding aY5 receptor. In one embodiment, the Y5 receptor is a mammalian receptor.In further embodiments, the Y5 receptor is a human, rat, or caninereceptor.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to mRNA encoding a Y5 receptor so asto prevent translation of the mRNA.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to the genomic DNA of a Y5 receptor.

This invention provides an antisense oligonucleotide of a Y5 receptorcomprising chemical analogues of nucleotides.

This invention further provides an antibody directed to a Y5 receptor.This invention also provides an antibody directed to a human Y5receptor.

This invention also provides a monoclonal antibody directed to anepitope of a human Y5 receptor present on the surface of a Y5 receptorexpressing cell.

Additionally, this invention provides a pharmaceutical compositioncomprising an amount of the oligonucleotide effective to reduce activityof a human Y5 receptor by passing through a cell membrane and bindingspecifically with mRNA encoding a human Y5 receptor in the cell so as toprevent its translation and a pharmaceutically acceptable carriercapable of passing through a cell membrane. In an embodiment, theoligonucleotide is coupled to a substance which inactivates mRNA. Inanother embodiment, the substance which inactivates mRNA is a ribozyme.

This invention further provides the above-described pharmaceuticalcomposition, wherein the pharmaceutically acceptable carrier capable ofpassing through a cell membrane comprises a structure which binds to areceptor specific for a selected cell type and is thereby taken up bycells of the selected cell type.

This invention additionally provides a pharmaceutical compositioncomprising an amount of an antagonist effective to reduce the activityof a human Y5 receptor and a pharmaceutically acceptable carrier. Thisinvention also provides a pharmaceutical composition comprising anamount of an agonist effective to increase activity of a Y5 receptor anda pharmaceutically acceptable carrier. This invention further provides apharmaceutical composition comprising and effective amount of a chemicalcompound identified by the above-described methods and apharmaceutically acceptable carrier. This invention also provides theabove-described pharmaceutical composition which comprises an amount ofthe antibody effective to block binding of a ligand to the Y5 receptorand a pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carriers” means any of thestandard pharmaceutically acceptable carriers. Examples include, but arenot limited to, phosphate buffered saline, physiological saline, waterand emulsions, such as oil/water emulsions.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a human Y5 receptor.

This invention provides a transgenic nonhuman mammal comprising ahomologous recombination knockout of the native Y5 receptor.

This invention provides a transgenic nonhuman mammal whose genomecomprises antisense DNA complementary to DNA encoding a human Y5receptor so placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding a Y5 receptor and which hybridizes tomRNA encoding a Y5 receptor thereby reducing its translation.

This invention provides the above-described transgenic nonhuman mammal,wherein the DNA encoding a human Y5 receptor additionally comprises aninducible promoter.

This invention provides the transgenic nonhuman mammal, wherein the DNAencoding a human Y5 receptor additionally comprises tissue specificregulatory elements.

In an embodiment, the transgenic nonhuman mammal is a mouse.

Animal model systems which elucidate the physiological and behavioralroles of Y5 receptor are produced by creating transgenic animals inwhich the activity of the Y5 receptor is either increased or decreased,or the amino acid sequence of the expressed Y5 receptor is altered, by avariety of techniques. Examples of these techniques include, but are notlimited to: 1) Insertion of normal or mutant versions of DNA encoding aY5 receptor, by microinjection, electroporation, retroviral transfectionor other means well known to those skilled in the art, into appropriatefertilized embryos in order to produce a transgenic animal or 2)Homologous recombination of mutant or normal, human or animal versionsof these genes with the native gene locus in transgenic animals to alterthe regulation of expression or the structure of these Y5 receptorsequences. The technique of homologous recombination is well known inthe art. It replaces the native gene with the inserted gene and so isuseful for producing an animal that cannot express native Y5 receptorsbut does express, for example, an inserted mutant Y5 receptor, which hasreplaced the native Y5 receptor in the animal's genome by recombination,resulting in underexpression of the transporter. Microinjection addsgenes to the genome, but does not remove them, and so is useful forproducing an animal which expresses its own and added Y5 receptors,resulting in overexpression of the Y5 receptors.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as M2 medium. DNA or cDNA encoding a Y5receptor is purified from a vector by methods well known in the art.Inducible promoters may be fused with the coding region of the DNA toprovide an experimental means to regulate expression of the trans-gene.Alternatively or in addition, tissue specific regulatory elements may befused with the coding region to permit tissue-specific expression of thetrans-gene. The DNA, in an appropriately buffered solution, is put intoa microinjection needle (which may be made from capillary tubing using apipet puller) and the egg to be injected is put in a depression slide.The needle is inserted into the pronucleus of the egg, and the DNAsolution is injected. The injected egg is then transferred into theoviduct of a pseudopregnant mouse (a mouse stimulated by the appropriatehormones to maintain pregnancy but which is not actually pregnant),where it proceeds to the uterus, implants, and develops to term. Asnoted above, microinjection is not the only method for inserting DNAinto the egg cell, and is used here only for exemplary purposes.

This invention also provides a method for determining whether a ligandcan specifically bind to a Y5 receptor which comprises contacting a celltransfected with and expressing DNA encoding the Y5 receptor, or amembrane fraction prepared from a cell extract of such cells, with theligand under conditions permitting binding of ligands to such receptor,detecting the presence of any such ligand specifically bound to the Y5receptor, and thereby determining whether the ligand specifically bindsto the Y5 receptor.

This invention provides a method for determining whether a ligand canspecifically bind to a Y5 receptor which comprises contacting a celltransfected with and expressing DNA encoding the Y5 receptor, or amembrane fraction from a cell extract of such cells, with the ligandunder conditions permitting binding of ligands to such receptor,detecting the presence of any such ligand specifically bound to the Y5receptor, and thereby determining whether the ligand specifically bindsto the Y5 receptor, wherein the Y5 receptor has substantially the sameamino acid sequence shown in FIG. 6.

This invention provides a method for determining whether a ligand canspecifically bind to a Y5 receptor which comprises contacting a celltransfected, with and expressing DNA encoding the Y5 receptor, or amembrane fraction of a cell extract of such cells, with the ligand underconditions permitting binding of ligands to such receptor, detecting thepresence of any such ligand specifically bound to the Y5 receptor, andthereby determining whether the ligand specifically binds to the Y5receptor, wherein the Y5 receptor is characterized by an amino acidsequence in the transmembrane region having 60% homology or higher tothe amino acid sequence in the transmembrane region of the Y5 receptorshown in FIG. 6.

In one embodiment of the above methods, the Y5 receptor is a human Y5receptor. In another embodiment of the above methods, the Y5 receptor isa rat Y5 receptor. In still another embodiment of the above methods, theY5 receptor is a canine Y5 receptor.

This invention provides a method for determining whether a ligand is aY5 receptor agonist which comprises contacting a cell transfected withand expressing a Y5 receptor, or a membrane frction from a cell extractof such cells, with the ligand under conditions permitting activation ofa functional Y5 receptor response, detecting a functional increase in Y5receptor activity, and thereby determining whether the ligand is a Y5receptor agonist. This invention further provides a method fordetermining whether a ligand is a Y5 receptor agonist which comprisescontacting a cell transfected with and expressing a Y5 receptor, or amembrane fraction prepared from a cell extract of such cells, with theligand under conditions permitting activation of the Y5 receptor,detecting an increase in Y5 receptor activity, and thereby determiningwhether the ligand is a Y5 receptor agonist.

In one embodiment of the above-described methods, the Y5 receptor is ahuman Y5 receptor. In another embodiment, the Y5 receptor is a rat Y5receptor. In a further embodiment, the Y5 receptor is a canine Y5receptor.

This invention also provides a method for determining whether a ligandis a Y5 receptor antagonist which comprises contacting a celltransfected with and expressing nucleic acid encoding a Y5 receptor, ora membrane fraction from a cell extract of such cells, with the ligandin the presence of a known Y5 receptor agonist, such as PYY or NPY,under conditions permitting the activation of a functional Y5 receptorresponse, detecting a decrease in Y5 receptor activity, and therebydetermining whether the ligand is a Y5 receptor antagonist. Thisinvention further provides a method for determining whether a ligand isa Y5 receptor antagonist which comprises contacting a cell transfectedwith and expressing DNA encoding a Y5 receptor, or a membrane fractionfrom a cell extract of such cells, with the ligand in the presence of aknown Y5 receptor agonist, such as PYY or NPY, under conditionspermitting the activation of the Y5 receptor, detecting a decrease in Y5receptor activity, and thereby determining whether the ligand is a Y5receptor antagonist.

In one embodiment of the above-described methods, the Y5 receptor is ahuman Y5 receptor. In another embodiment, the Y5 receptor is a rat Y5receptor. In a further embodiment, the Y5 receptor is a canine Y5receptor. In an embodiment of the methods described hereinabove andhereinbelow, the cell is a Xenopus cell such as an oocyte or melanophorecell. In another embodiment of the methods described herein, the cell isa neuronal cell such as the glial cell line C6. In yet anotherembodiment of the methods described herein, the cell is non-neuronal inorigin. In a further embodiment, the non-neuronal cell is a COS-7 cell,CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk-) cell.In still further embodiments of the methods described herein, the cellmay be an insect cell such as a Sf-9 cell or Sf-21 cell. In oneembodiment of the above-described methods, the ligand is not previouslyknown.

This invention additionally provides a Y5 receptor agonist detected bythe above-described method. This invention also provides a Y5 receptorantagonist detected by the above-described method.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a Y5 receptor to identify a compoundwhich specifically binds to the Y5 receptor which comprises (a)contacting a cell transfected with and expressing DNA encoding the Y5receptor, or a membrane fraction from a cell extract of such cells, witha compound known to bind specifically to the Y5 receptor; (b) contactingthe preparation of step (a) with the plurality of compounds not known tobind specifically to the Y5 receptor, under conditions permittingbinding of compounds known to bind to the Y5 receptor; (c) determiningwhether the binding of the compound known to bind to the Y5 receptor isreduced in the presence of the compounds, relative to the binding of thecompound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the Y5 receptor of each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which specifically binds to the Y5 receptor.

Such competitive binding assays provide an efficient means to assess thereceptor binding of chemical compounds either singly or in mixtures suchas may be present in extracts of natural products or generated usingcombinatorial chemical synthetic methods for the production of peptidyland non-peptidyl compounds.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a Y5 receptor to identify a compoundwhich activates the Y5 receptor which comprises (a) contacting a celltransfected with and expressing the Y5 receptor, or with a membranefraction from a cell extract of such cells, with the plurality ofcompounds not known to bind specifically to the Y5 receptor, underconditions permitting activation of the Y5 receptor, (b) determiningwhether the activity of the Y5 receptor is increased in the presence ofthe compounds; and if so (c) separately determining whether theactivation of the Y5 receptor is increased by each compound included inthe plurality of compounds, so as to thereby identify the compound whichactivates the Y5 receptor.

This invention further provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a Y5 receptorto identify a compound which inhibits the activation of the Y5 receptor,which comprises (a) contacting a cell transfected with and expressingthe Y5 receptor, or a membrane fraction from a cell extract of suchcells, with the plurality of compounds in the presence of a known Y5receptor agonist, under conditions permitting activation of the Y5receptor; (b) determining whether the activation of the Y5 receptor isreduced in the presence of the plurality of compounds, relative to theactivation of the Y5 receptor in the absence of the plurality ofcompounds; and if so (c) separately determining the inhibition ofactivation of the Y5 receptor for each compound included in theplurality of compounds, so as to thereby identify the compound whichinhibits the activation of the Y5 receptor.

In one embodiment of the above-described methods the Y5 receptor is ahuman Y5 receptor. In another embodiment, the Y5 receptor is a rat Y5receptor. In a further embodiment, the Y5 receptor is a canine Y5receptor. In an embodiment of the methods described herein, the cell isa Xenopus cell such as an oocyte or melanophore cell. In anotherembodiment, the cell is a mammalian cell. In a further embodiment, themammalian cell is non-neuronal in origin. The cell may be a COS-7 cell,CHO cell, a 293 human embryonic kidney cell, a LM(tk-) cell, or anNIH-3T3 cell. In still further embodiments, the cell is an insect cellsuch as a Sf-9 cell, Sf-21 cell, or HighFive cell.

Additionally, this invention provides a method of screening drugs toidentify drugs which specifically bind to a Y5 receptor on the surfaceof a cell which comprises contacting a cell transfected with andexpressing DNA encoding a Y5 receptor, or a membrane fraction from acell extract of such cells, with a plurality of drugs under conditionspermitting binding of drugs to the Y5 receptor, determining those drugswhich specifically bind to the transfected cell, and thereby identifyingdrugs which specifically bind to the Y5 receptor.

This invention provides a method of screening drugs to identify drugswhich act as agonists of a Y5 receptor which comprises contacting a celltransfected with and expressing DNA encoding a Y5 receptor with aplurality of drugs under conditions permitting the activation of afunctional Y5 receptor response, determining those drugs which activatesuch receptor in the cell, and thereby identify drugs which act as Y5receptor agonists.

This invention provides a method of screening drugs to identify drugswhich act as Y5 receptor antagonists which comprises contacting cellstransfected with and expressing DNA encoding a Y5 receptor, or amembrane fraction from a cell extract of such cells, with a plurality ofdrugs in the presence of a known Y5 receptor agonist, such as PYY orNPY, under conditions permitting the activation of a functional Y5receptor response, determining those drugs which inhibit the activationof the receptor in the mammalian cell, and thereby identifying drugswhich act as Y5 receptor antagonists. In one embodiment of theabove-described methods, the cell is a mammalian cell. In anotherembodiment, the cell is nonneuronal in origin.

This invention also provides a process for identifying a chemicalcompound which specifically binds to a Y5 receptor, which comprisescontacting nonneuronal cells expressing on their cell surface the Y5receptor, or a membrane fraction from a cell extract of such cells, withthe chemical compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the Y5 receptor.

This invention further provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to a Y5receptor which comprises separately contacting nonneuronal cellsexpressing on their cell surface a Y5 receptor, or a membrane fractionfrom a cell extract of such cells, with both the chemical compound and asecond chemical compound known to bind to the receptor, and with onlythe second chemical compound, under conditions suitable for binding ofboth compounds, and detecting specific binding of the chemical compoundto the Y5 receptor, a decrease in the binding of the second chemicalcompound to the Y5 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the Y5 receptor.

This invention additionally provides a process for determining whether achemical compound specifically binds to and activates a Y5 receptor,which comprises contacting nonneuronal cells producing a secondmessenger response and expressing on their cell surface a Y5 receptor,or a membrane fraction from a cell extract of such cells, with thechemical compound under conditions suitable for activation of the Y5receptor, and measuring the second messenger response in the presenceand in the absence of the chemical compound, a change in secondmessenger response in the presence of the chemical compound indicatingthat the chemical compound activates the Y5 receptor.

This invention also provides a process for determining whether achemical compound specifically binds to and inhibits activation of a Y5receptor, which comprises separately contacting nonneuronal cellsproducing a second messenger response and expressing on their cellsurface a Y5 receptor, or a membrane fraction from a cell extract ofsuch cells, with both the chemical compound and a second chemicalcompound known to activate the Y5 receptor, and with only the secondchemical compound, under conditions suitable for activation of the Y5receptor, and measuring the second messenger response in the presence ofonly the second chemical compound and in the presence of both thechemical compound and the second chemical compound, a smaller change insecond messenger response in the presence of both the chemical compoundand the second chemical compound indicating that the chemical compoundinhibits activation of the Y5 receptor.

In one embodiment of the above-described methods, the second messengercomprises adenylate cyclase activity and the change in second messengerresponse is a decrease in adenylate cyclase activity. In a furtherembodiment, the second messenger response comprises adenylate cyclaseactivity and the change in second messenger response is a smallerdecrease in the level of adenylate cyclase activity in the presence ofboth the chemical compound and the second chemical compound than in thepresence of only the second chemical compound. In another is embodiment,the second messenger comprises intracellular calcium levels and thechange in second messenger response is an increase in intracellularcalcium levels. In a further embodiment, the second messenger comprisesintracellular calcium levels and the change in second messenger responseis a smaller increase in the level of intracellular calcium in thepresence of both the chemical compound and the second chemical compoundthan in the presence of only the second chemical compound.

In an embodiment of any of the above-described methods, the cell is amammalian cell. In a further embodiment, the cell is a COS-7 cell, a 293human embryonic kidney cell, an LM(tk-) cell or an NIH-3T3 cell. It isfurther to be understood that any of the cells described herein, or anyother appropriate host cell, may be used to express the Y5 receptors ofthe subject invention in any of the above-described embodiments. In oneembodiment, the Y5 receptor is a human Y5 receptor. In furtherembodiments, the Y5 receptor is a rat or a canine Y5 receptor.

The binding and functional assays described herein may be performedusing any cells which express the Y5 receptors of the subject invention,including, but not limited to, cells transfected with exogenous nucleicacid encoding Y5 receptors, as well as cultured cells or cell linescultured under conditions which lead to expression of Y5 receptorsdetectable by either binding or functional assays.

This invention also provides for any of the above methods fordetermining whether a compound activates or inhibits activation of anyof the Y5 receptors described herein, wherein the activation isdetermined not by means of a second messenger response, but by effectsof receptor activation which may occur prior to or independent of asecond messenger response. In an embodiment, measurement of the secondmessenger response is replaced with measurement of a change in thebinding of GTPγS (a non-hydrolyzable analog of GTP) to cells transfectedwith and expressing a Y5 receptor or to a membrane fraction from suchcells. Preferably, the cells are nonneuronal cells. In a furtherembodiment, an increase in GTPγS binding to the cells or the membranefraction in the presence of a compound indicates that the compoundactivates the Y5 receptor. In yet another embodiment, a smaller increasein GTPγS binding to the cells or membrane fraction in the presence ofboth a compound known to activate the receptor and a test compound,relative to the increase in GTPγS binding in the presence of only thecompound known to activate the receptor, indicates that the testcompound inhibits activation of the Y5 receptor. In other embodiments,activation or inhibition of activation of any of the Y5 receptorsdisclosed herein may be measured by other means not requiring a secondmessenger, such as activation of MAP kinase, or activation of a reportergene system, or by activation of immediate early genes, which are wellknown in the art.

This invention provides a process for determining whether a chemicalcompound specifically binds to and activates a Y5 receptor, whichcomprises contacting nonneuronal cells expressing a Y5 receptor, or amembrane fraction from a cell extract of such cells, with the chemicalcompound under conditions suitable for activation of the Y5 receptor,and measuring the binding of GTPγS to the cells or membrane fraction, inthe presence and in the absence of the chemical compound, a change inthe binding of GTPγS in the presence of the chemical compound indicatingthat the chemical compound activates the Y5 receptor. This inventionfurther provides a process for determining whether a chemical compoundspecifically binds to and inhibits activation of a Y5 receptor, whichcomprises separately contacting nonneuronal cells expressing a Y5receptor, with both the chemical compound and a second chemical compoundknown to activate the Y5 receptor, and with only the second chemicalcompound, under conditions suitable for activation of the Y5 receptor,and measuring binding of GTPγS to the cell or membrane fraction in thepresence of only the second chemical compound and in the presence ofboth the second chemical compound and the chemical compound, a smallerchange in GTPγS binding in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of a Y5 receptor. In one embodiment of the above-describedmethods the change in binding is an increase in GTPγS binding. Inanother embodiment, the change in binding is a smaller increase in GTPγSbinding in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound. In another embodiment, the cells are not intact.

It is known in the art that that in cell lines, the expression level ofendogenous receptors can be increased several-fold by treatment withcompounds such as I1-1β (Menke, et al., 1994), NGF (Dimaggio, et al.,1994) or glucocorticoids (Larsen, et al., 1994). Such treatment mayallow screening of compounds at Y5 receptors in cell lines expressingpreviously undetectable levels of endogenous Y5 receptors, withouttransfecting such cell lines with the Y5 receptor. One may also createrecombinant cell lines, whereby the normal promoter may be replaced withpromoter element(s) that allow increased expression of the Y5 gene,thereby allowing one to screen compounds using the recombinant cellline. Such cells and cell lines may be used with any of theabove-described methods or processes.

This invention provides a pharmaceutical composition comprising a drugidentified by the above-described methods and a pharmaceuticallyacceptable carrier.

This invention provides a method of detecting expression of Y5 receptorby detecting the presence of mRNA coding for the Y5 receptor whichcomprises obtaining total mRNA from the cell and contacting the mRNA soobtained with the above-described nucleic acid probe under hybridizingconditions, detecting the presence of mRNA hybridized to the probe, andthereby detecting the expression of the Y5 receptor by the cell.

This invention provides a method of treating obesity and other disordersassociated with excess eating (e.g., bulimia) in which a Y5 receptorantagonist is administered in combination with existing therapies. Anexample os such a drug is dexfenfluramine, a serotonin uptake inhibitor(McTavish, D. and R. C. Heel, Drugs 43(5):713-733 (1992)).Administration of dexfenfluramine results in significant weight lossafter about one month of therapy, with maximal weight loss occurring inthe first six months of therapy. It is noteworthy that afterdiscontinuation of drug therapy an increase in body weight is observedafter about two months. On e study reports that no statisticallysignificant differences from placebo were observable by five monthsafter discontinuing drug therapy (O'Connor, H. T. et al., Int. J. Obes.Relat. Metab. Disord. 19(3):30-337 (1991)). Although the potentialusefulness of sibutramine therapy has not been fully explored,combinations of sibutramine and a Y5 receptor antagonist may also proveuseful.

This invention provides a method of decreasing feeding behavior in asubject which comprises administering to the subject a compound which isa Y5 receptor antagonist and a compound which is monoamineneurotransmitter uptake inhibitor, wherein the amount of the Y5antagonist and the monoamine neurotransmitter uptake inhibitor areeffective to decrease the feeding behavior of the subject. Thisinvention also provides the use of a compound which is a Y5 receptorantagonist and a compound which is a monoamine neurotransmitter uptakeinhibitor for the preparation of a pharmaceutical composition fordecreasing feeding behavior in a subject, wherein the amount of the Y5receptor antagonist and the amount of the monoamine neurotransmitteruptake inhibitor is effective to decrease feeding behavior in thesubject. In one embodiment of the above-described methods, the Y5receptor antagonist and the monoamine neurotransmitter uptake inhibitorare administered in combination. In another embodiment, the Y5 receptorantagonist and the monoamine neurotransmitter uptake inhibitor areadministered once. In a further embodiment, the Y5 receptor antagonistand the, monoamine neurotransmitter uptake inhibitor are administeredseparately. In another embodiment, the Y5 receptor antagonist and themonoamine neurotransmitter uptake inhibitor are administered once. Inone embodiment, the Y5 receptor antagonist is administered for about twoweeks to about six months. In another embodiment, the monoamineneurotransmitter uptake inhibitor is administered for about one month toabout six months. In a further embodiment, the Y5 receptor antagonistand the monoamine neurotransmitter uptake inhibitor are administeredrepeatedly. In another embodiment, the Y5 receptor antagonist isadministered for about two weeks to about six months. In one embodiment,the monoamine neurotransmitter uptake inhibitor is administered forabout one month to about six months. In another embodiment, theneurotransmitter uptake inhibitor is administered for about one month toabout three months. In separate embodiments, the monoamineneurotransmitter uptake inhibitor may be fenfluramine, dexfenfluramine,or sibutramine. In another embodiment, the compound is administered in apharmaceutical composition comprising a sustained release formula.

This invention provides a method of decreasing feeding behavior of asubject which comprises administering to the subject a compound which isa galanin receptor antagonist and a compound which is a Y5 receptorantagonist, wherein the amount of the antagonists is effective todecrease feeding behavior of the subject. In one embodiment, the galaninreceptor antagonist and the Y5 receptor antagonist are administered incombination. In another embodiment the galanin receptor antagonist andthe Y5 receptor antagonist are administered once. In a furtherembodiment the galanin receptor antagonist and the Y5 receptorantagonist are administered separately. In another embodiment thegalanin receptor antagonist and the Y5 receptor antagonist areadministered once. In an embodiment the galanin receptor antagonist isadministered for about 1 week to about 2 weeks. In a further embodimentthe Y5 receptor antagonist is administered for about 1 week to about 2weeks. In another embodiment, the galanin receptor antagonist and the Y5receptor antagonist are administered repeatedly. In an embodiment, thegalanin receptor antagonist is administered for about 1 week to about 2weeks. In separate embodiments, the galanin receptor is a GALR2 receptoror a GALR3 receptor. In another embodiment the compound is administeredin a pharmaceutical composition comprising a sustained releaseformulation.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition of a Y5receptor which comprises administering to a subject an amount of theabove-described pharmaceutical composition effective to decrease theactivity of the Y5 receptor in the subject and thereby treat theabnormality.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by the --activation of a Y5receptor which comprises administering to a subject an amount of theabove-described pharmaceutical composition effective to increase theactivation of the Y5 receptor in the subject and thereby treat theabnormality.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the decreasing theactivity of a Y5 receptor which comprises administering to a subject anamount of the above-described pharmaceutical composition effective todecrease the activity of the Y5 receptor and thereby treat theabnormality.

In one embodiment of the above-described methods, the abnormality isobesity. In another embodiment, the abnormality is bulimia.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by the activation of a Y5 receptorwhich comprises administering to a subject an effective amount of a Y5receptor agonist. In a further embodiment, the abnormal condition isanorexia. In a separate embodiment, the abnormal condition is asexual/reproductive disorder. In another embodiment, the abnormalcondition is depression. In another embodiment, the abnormal conditionis anxiety.

In an embodiment, the abnormal condition is gastric ulcer. In a furtherembodiment, the abnormal condition is memory loss. In a furtherembodiment, the abnormal condition is migraine. In a further embodiment,the abnormal condition is pain. In a further embodiment, the abnormalcondition is epileptic seizure. In a further embodiment, the abnormalcondition is hypertension. In a further embodiment, the abnormalcondition is cerebral hemorrhage. In a further embodiment, the abnormalcondition is shock. In a further embodiment, the abnormal condition iscongestive heart failure. In a further embodiment, the abnormalcondition is sleep disturbance. In a further embodiment, the abnormalcondition is nasal congestion. In a further embodiment, the abnormalcondition is diarrhea.

This invention further provides a method of treating obesity in asubject which comprises administering to the subject an effective amountof a Y5 receptor antagonist. This invention also provides a method oftreating anorexia in a subject which comprises administering to thesubject an effective amount of a Y5 receptor agonist.

In addition, this invention provides a method of treating bulimianervosa in a subject which comprises administering to the subject aneffective amount of a Y5 receptor antagonist.

This invention provides a method of inducing a subject to eat whichcomprises administering to the subject an effective amount of a Y5receptor agonist. In one embodiment, the subject is a vertebrate. Inanother embodiment, the subject is a human. In another embodiment, thesubject is a rat. In another embodiment, the subject is a caninesubject. This invention also provides a method of increasing theconsumption of a food product by a subject which comprises administeringto the subject a composition of the food product and an amount of a Y5receptor agonist. In one embodiment, the subject is a vertebrate. Inanother embodiment, the subject is a human, a rat or a canine subject.

This invention also provides a method of treating abnormalities whichare alleviated by reduction of activity of a human Y5 receptor whichcomprises administering to a subject an amount of the above-describedpharmaceutical composition effective to reduce the activity of human Y5receptor and thereby alleviate abnormalities resulting from overactivityof a human Y5 receptor. This invention further provides a method oftreating an abnormal condition related to an excess of Y5 receptoractivity which comprises administering to a subject an amount of thepharmaceutical composition effective to block binding of a ligand to theY5 receptor and thereby alleviate the abnormal condition.

This invention additionally provides a method of detecting the presenceof a Y5 receptor on the surface of a cell which comprises contacting thecell with the antibody capable of binding to the Y5 receptor underconditions permitting binding of the antibody to the receptor, detectingthe presence of the antibody bound to the cell, and thereby detectingthe presence of a Y5 receptor on the surface of the cell.

This invention also provides a method of determining the physiologicaleffects of varying levels of activity of a Y5 receptor which comprisesproducing a transgenic nonhuman mammal whose levels of Y5 receptoractivity are varied by use of an inducible promoter which regulates Y5receptor expression. This invention further provides a method ofdetermining the physiological effects of varying levels of activity of aY5 receptors which comprises producing a panel of transgenic nonhumanmammals each expressing a different amount of Y5 receptor.

This invention provides a method for identifying a substance capable ofalleviating the abnormalities resulting from overactivity of a Y5receptor comprising administering a substance to the above-describedtransgenic nonhuman mammals, and determining whether the substancealleviates the physical and behavioral abnormalities displayed by thetransgenic nonhuman mammal as a result of overactivity of a Y5 receptor.

This invention also provides a method for treating abnormalitiesresulting from overactivity of a Y5 receptor which comprisesadministering to a subject an amount of the above-describedpharmaceutical composition effective reduce the activation of the Y5receptor and thereby alleviate the abnormalities resulting fromoveractivity of a Y5 receptor.

This invention further provides a method for identifying a substancecapable of alleviating the abnormalities resulting from underactivity ofa Y5 receptor comprising administering the substance to theabove-described transgenic nonhuman mammals and determining whether thesubstance alleviates the physical and behavioral abnormalities displayedby the transgenic nonhuman mammal as a result of underactivity of a Y5receptor.

This invention additionally provides a method for treating theabnormalities resulting from underactivity of a Y5 receptor whichcomprises administering to a subject an amount of the above-describedpharmaceutical composition effective to increase the activation of theY5 receptor and thereby alleviate the abnormalities resulting fromunderactivity of a Y5 receptor.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific allelic form of a Y5receptor which comprises: a. obtaining DNA from the subject to betested; digesting the DNA with restriction enzymes; c. separating theresulting DNA fragments; d. contacting the fragments with a detectablylabeled nucleic acid probe capable of specifically hybridizing with asequence uniquely present within the sequence of a nucleic acid encodingthe allelic form of the Y5 receptor; and e. detecting the presence oflabeled probe hybridized to the DNA fragments from the subject beingtested, the presence of such hybridized probe indicating that thesubject is predisposed to the disorder.

This invention also provides a method of preparing an isolated Y5receptor which comprises: a. inducing cells to express the Y5 receptor;b. recovering the receptor from the resulting cells; and c. purifyingthe receptor so recovered. This invention further provides a method ofpreparing the isolated Y5 receptor which comprises: a. inserting nucleicacid encoding Y5 receptor in a suitable vector adapted for expression ina bacterial, yeast, insect, or mammalian cell operatively linked to thenucleic acid encoding the Y5 receptor as to permit expression thereof;b. inserting the resulting vector in a suitable host cell so as toobtain a cell which produces the Y5 receptor; c. recovering the receptorproduced by the resulting cell; and d. purifying the receptor sorecovered.

This invention provides a method for detecting in a subject the presenceof a restriction fragment length polymorphism associated with a genomiclocus which encompasses both a Y1 and a Y5 receptor gene whichcomprises: a) obtaining a sample of DNA from the subject; b) digestingthe DNA with a restriction, enzyme; c) separating the resulting DNAfragments; d) contacting the DNA fragments with a detectably labelednucleic acid probe which specifically hybridizes with a sequenceuniquely present within the sequence associated with the polymorphism;and e) detecting whether the probe hybridizes to the DNA fragments, thepresence of the labeled probe hybridized to the DNA fragment indicatingthe presence of the restriction fragment length polymorphism.

In an embodiment of the above-described method, the restriction enzymeis PstI. In another embodiment, the subject is a human. In still anotherembodiment, the PstI polymorphism is associated with susceptibility tomodification of feeding behavior using a Y5-selective compound. Invarious embodiments, the feeding behavior is anorexia or bulimia, or thefeeding behavior is associated with obesity.

In an embodiment of any of the above-described methods, the subject is ahuman. In another embodiment, the subject is a non-human animal. Instill another embodiment, the subject is a mammal. In yet anotherembodiment, the subject is a bovine, equine, canine or feline.

This invention provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non-peptidylcompound which is a Y5 receptor antagonist in an amount effective toinhibit the activity of the subject's Y5 receptor, wherein the bindingof the compound to the human Y5 receptor is characterized by a K_(i)less than 100 nanomolar when measured in the presence of ¹²⁵I-PYY at apredetermined concentration, and wherein the compound binds to the humanY5 receptor with an affinity greater than ten-fold higher than theaffinity with which the compound binds to any other human Y-typereceptor.

In an embodiment of the above-described method, the binding of thecompound to each of the human Y1, human Y2, and human Y4 receptors ischaracterized by a K_(i) greater than 500 nanomolar when measured in thepresence of ¹²⁵I-PYY at a predetermined concentration. In anotherembodiment, the binding of the compound to each of the human Y1, humanY2, and human Y4 receptors is characterized by a K_(i) greater than 1000nanomolar.

This invention also provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non-peptidylcompound which is a Y5 receptor antagonist in an amount effective toinhibit the activity of the subject's Y5 receptor, wherein the bindingof the compound to the human Y5 receptor is characterized by a K_(i)less than 5 nanomolar when measured in the presence of ¹²⁵I-PYY at apredetermined concentration.

In an embodiment of the above-described method, the compound to each ofthe human Y1, human Y2, and human Y4 receptors is characterized by aK_(i) greater than 5 nanomolar when measured in the presence of 125I-PYYat a predetermined concentration. In another embodiment of theabove-described method, the compound binds to the human Y5 receptor withan affinity greater than ten-fold higher than the affinity with whichthe compound binds to any other human Y-type receptor. In yet anotherembodiment, the binding of the compound to each of the human Y1, humanY2 and human Y4 receptors is characterized by a K_(i) greater than 50nanomolar when measured in the presence of ¹²⁵I-PYY at a predeterminedconcentration. In still another embodiment, the binding of the compoundto each of the human Y1, human Y2 and human Y4 receptors ischaracterized by a K_(i) greater than 100 nanomolar.

This invention further provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non-peptidylcompound which is a Y5 receptor antagonist in an amount effective toinhibit the activity of the subject's Y5 receptor, wherein the compoundbinds to the human Y5 receptor with an affinity greater than ten-foldhigher than the affinity with which the compound binds to any otherhuman Y-type receptor. In an embodiment of the above-described method,the compound binds to the human Y5 receptor with an affinity greaterthan ten-fold higher than the affinity with which the compound binds toany other human Y-type receptor, and greater than 26-fold higher thanthe affinity with which the compound binds to the human Y1 receptor. Inanother embodiment of the above-described methods, the compound binds tothe human Y5 receptor with an affinity greater than ten-fold higher thanthe affinity with which the compound binds to any other human Y-typereceptor, and greater than 22-fold higher than the affinity with whichthe compound binds to the human Y2 receptor. In still another embodimentof the above-described methods, the compound binds to the human Y5receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to any other human Y-type receptor, andgreater than 34-fold higher than the affinity with which the compoundbinds to the human Y4 receptor.

In another embodiment of the above-described methods, the compound bindsto the human Y5 receptor with an affinity a) greater than ten-foldhigher than the affinity with which the compound binds to any otherhuman Y-type receptor; b) greater than 22-fold higher than the affinitywith which the compound binds to the human Y2 receptor; and c) greaterthan 34-fold higher than the affinity with which the compound binds tothe human Y4 receptor. In another embodiment of the above-describedmethods, the compound binds to the human Y5 receptor with an affinitygreater than ten-fold higher than the affinity with which the compoundbinds to any other human Y-type receptor, and with an affinity a)greater than 26-fold higher than the affinity with which the compoundbinds to the human Y1 receptor; b) greater than 22-fold higher than theaffinity with which the compound binds to the human Y2 receptor; and c)and greater than 34-fold higher than the affinity with which thecompound binds to the human Y4 receptor. In yet another embodiment ofthe above described methods, the compound binds to the human Y5 receptorwith an affinity greater than ten-fold higher than the affinity withwhich the compound binds to any other human Y-type receptor, and greaterthan 100-fold higher than the affinity with which the compound binds tothe human Y1 receptor. In a further embodiment of the above describedmethods, the compound binds to the human Y5 receptor with an affinitygreater than ten-fold higher than the affinity with which the compoundbinds to any other human Y-type receptor, and greater than 165-foldhigher than the affinity with which the compound binds to the human Y2receptor.

In another embodiment of the above described methods, the compound bindsto the human Y5 receptor with an affinity greater than ten-fold higherthan the affinity with which the compound binds to any other humanY-type receptor, and greater than 143-fold higher than the affinity withwhich the compound binds to the human Y4 receptor. In yet anotherembodiment of the above described methods, the compound binds to thehuman Y5 receptor with an affinity greater than ten-fold higher than theaffinity with which the compound binds to any other human Y-typereceptor and a) greater than 143-fold higher than the affinity withwhich the compound binds to the human Y4 receptor; and b) greater than165-fold higher than the affinity with which the compound binds to thehuman Y2 receptor. In still yet another embodiment of the abovedescribed methods, the compound binds to the human Y5 receptor with anaffinity greater than ten-fold higher than the affinity with which thecompound binds to any other human Y-type receptor, and a) greater than143-fold higher than the affinity with which the compound binds to thehuman Y4 receptor; b) greater than 165-fold higher than the affinitywith which the compound binds to the human Y2 receptor; and c) greaterthan 100-fold higher than the affinity with which the compound binds tothe human Y1 receptor.

This invention additionally provides a method of treating a subject'sfeeding disorder which comprises administering to the subject anon-peptidyl compound which is a Y5 receptor antagonist in an amounteffective to inhibit the activity of the subject's Y5 receptor, whereinthe compound binds to the human Y5 receptor with an affinity greaterthan 500-fold higher than the affinity with which the compound binds toeach of the human Y1, human Y2, and human Y4 receptors.

This invention also provides a method of treating a subject's feedingdisorder which comprises administering to the subject a non-peptidylcompound which is a Y5 receptor antagonist in an amount effective toinhibit the activity of the subject's Y5 receptor, wherein the compoundbinds to the human Y5 receptor with an affinity greater than 1400-foldhigher than the affinity with which the compound binds to each of thehuman Y1, human Y2, and human Y4 receptors.

In an embodiment of any of the above methods, the feeding disorder isobesity or bulimia. In a further embodiment of any of the above methods,the subject is a vertebrate, a mammal, a human or a canine.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS Materials and Methods cDNA Cloning

Total RNA was prepared by a modification of the guanidine thiocyanatemethod (Kingston, 1987), from 5 grams of rat hypothalamus (Rockland,Gilbertsville, Pa.). Poly A⁺RNA was purified with a FastTrack kit(Invitrogen Corp., San Diego, Calif.). Double stranded (ds) cDNA wassynthesized from 7 μg of poly A⁺ RNA according to Gubler and Hoffman(Gubler and Hoffman, 1983), except that ligase was omitted in the secondstrand cDNA synthesis. The resulting ds-cDNA was ligated to BstXI/EcoRIadaptors (Invitrogen Corp.), the excess of adaptors was removed bychromatography on Sephacryl 500 HR (Pharmacia®-LKB) and the ds-cDNA sizeselected on a Gen-Pak Fax HPLC column (Millipore Corp., Milford, Mass.).High molecular weight fractions were ligated in pEXJ.BS (A cDNA cloningexpression vector derived from pcEXV-3; Okayama and Berg, 1983; Millerand Germain, 1986) cut by BstXI as described by Aruffo and Seed (Aruffoand Seed, 1987). The ligated DNA was electroporated in E. coli MC 1061F⁺ (Gene Pulser, Biorad). A total of 3.4×10⁶ independent clones with aninsert mean size of 2.7 kb could be generated. The library was plated onPetri dishes (Ampicillin selection) in pools of 6.9 to 8.2×10³independent clones. After 18 hours amplification, the bacteria from eachpool were scraped, resuspended in 4 mL of LB media and 1.5 mL processedfor plasmid purification with a QIAprep-8 plasmid kit (Qiagen Inc,Chatsworth, Calif.). 1 ml aliquots of each bacterial pool were stored at−85° C. in 20% glycerol.

Isolation of a cDNA Clone Encoding an Atypical Rat Hypothalamic NPY5Receptor

DNA from pools of ≈7500 independent clones was transfected into COS-7cells by a modification of the DEAE-dextran procedure (Warden andThorne, 1968). COS-7 cells were grown in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml ofpenicillin, 100 μg/ml of streptomycin, 2 mM L-glutamine (DMEM-C) at 37°C. in 5% CO₂. The cells were seeded one day before transfection at adensity of 30,000 cells/cm² on Lab-Tek chamber slides (1 chamber,Permanox slide from Nunc Inc., Naperville, Ill.). On the next day, cellswere washed twice with PBS, 735 μl of transfection cocktail was addedcontaining 1/10 of the DNA from each pool and DEAE-dextran (500 μg/ml)in Opti-MEM I serum free media (Gibco BRL LifeTechnologies Inc. GrandIsland, N.Y.). After a 30 min. incubation at 37° C., 3 ml of chloroquine(80 μM in DMEM-C) was added and the cells incubated a further 2.5 hoursat 37° C. The media was aspirated from each chamber and 2 ml of 10% DMSOin DMEM-C added. After 2.5 minutes incubation at room temperature, themedia was aspirated, each chamber washed once with 2 ml PBS, the cellsincubated 48 hours in DMEM-C and the binding assay was performed on theslides. After one wash with PBS, positive pools were identified byincubating the cells with 1 nM (3×10⁶ cpm per slide) of porcine[¹²⁵I]-PYY (NEN; SA=2200 Ci/mmole) in 20 mM Hepes-NaOH pH 7.4, CaCl₂1.26 mM, MgSO₄ 0.81 mM, KH²PO₄ 0.44 mM, KCL 5.4, NaCl 10 mM, 0.1% BSA,0.1% bacitracin for 1 hour at room temperature. After six washes (threeseconds each) in binding buffer without ligand, the monolayers werefixed in 2.5% glutaraldehyde in PBS for five minutes, washed twice fortwo minutes in PBS, dehydrated in ethanol baths for two minutes each(70, 80, 95, 100%) and air dried. The slides were then dipped in 100%photoemulsion (Kodak type NTB2) at 42° C. and exposed in the dark for 48hours at 4° C. in light proof boxes containing drierite. Slides weredeveloped for three minutes in Kodak D19 developer (32 g/L of water),rinsed in water, fixed in Kodak fixer for 5 minutes, rinsed in water,air dried and mounted with Aqua-Mount (Lerner Laboratories, Pittsburgh,Pa.). Slides were screened at 25×total magnification. A single clone,CG-18, was isolated by SIB selection as described (Mc Cormick, 1987).DS-DNA was sequenced with a Sequenase kit (US Biochemical, Cleveland,Ohio) according to the manufacturer. Nucleotide and peptide sequenceanalysis were performed with GCG programs (Genetics Computer Group,Madison, Wis.).

Isolation of the Human Y5 Homolog

Using rat oligonucleotide primers in TM 3 (sense primer; position484-509 in FIG. 1A) and in TM 6 (antisense primer; position 1219-1243 inFIG. 3A), applicants screened a human hippocampal cDNA library using thepolymerase chain reaction. 1 μl (4×10⁶ bacteria) of each of 450amplified pools containing each ≈5000 independent clones andrepresenting a total of 2.2×10⁶ was subjected directly to 40 cycles ofPCR and the resulting products analyzed by agarose gel electrophoresis.One of three positive pools was analyzed further and by sib selection asingle cDNA clone was isolated and characterized. This cDNA turned outto be full length and in the correct orientation for expression. DS-DNAwas sequenced with a sequenase kit (US Biochemical, Cleveland, Ohio)according to the manufacturer.

Isolation of the Canine Y5 Homolog

An alignment of the coding nucleotide sequences of the rat and human Y5receptors was used to synthesize a pair of PCR primers. A regionupstream of TM III which is 100% conserved between rat and human waschosen to synthesize the forward primer CH 156:

5′-TGGATCAGTGGATGTTTGGCAAAG-3′ (Seq. I.D. No. 7).

A region at the carboxy end of the 5-6 loop, immediately upstream ofTM6, which is also 100% conserved between rat and human sequences waschosen to synthesize the reverse primer CH153:

5′-GTCTGTAGAAAACACTTCGAGATCTCTT-3′ (Seq. I.D. No. 8).

The primers CH156-CH153 were used to amplify 10 ng of poly (A+) RNA fromrat brain that was reverse transcribed using the SSII reversetranscriptase (GibcoBRL, Gaithersburg, Md.). PCR was performed onsingle-stranded cDNA with Taq Polymerase (Perkin Elmer-Roche MolecularSystems, Branchburg, N.J.) under the following conditions: 94° C. for 1min, 60° C. for 1 min and 72° C. for 1 min for 40 cycles. The resulting798 bp PCR DNA fragment was subcloned in pCR Script (Stratagene, LaJolla, Calif.) and sequenced using a sequenase kit (USB, Cleveland,Ohio) and is designated Y5-bd-5.

3′ and 5′ RACE

It was attempted to isolate the missing 3′ and 5′ ends of the beagle dogY5 receptor sequences by 3′ and 5′ RACE using a Marathon cDNAamplification kit (Clontech, Palo Alto, Calif.). From the sequence ofthe canine (beagle) PCR DNA fragment described above, the following PCRprimers were synthesized:

(3′ RACE)

CH 204:

5′-CTTCCAGTGTTTCACAGTCTGGTGG-3′ (Seq. I.D. No. 9);

CH 218 (nested primer):

5′-CTGAGCAGCAGGTATTTATGTGTTG-3′ (Seq. I.D. No. 10);

(5′ RACE)

CH 219:

5′-CTGGATGAAGAATGCTGACTTCTTACAG-3′ (Seq. I.D. No. 11);

CH 245 (nested primer):

 5′-TTCTTGAGTGGTTCTCTTGAGGAGG-3′ (Seq. I.D. No. 12).

The 3′-and 5′ RACE reactions were carried out on canine thalamic cDNAaccording to the kit specifications, with the primers described above.The resulting PCR DNA products (smear of 0.7 to 10 kb) were purifiedfrom an agarose gel and reamplified using the nested primers describedabove. The resulting discrete DNA bands were again purified from anagarose gel and subcloned in pCR Script (Stratagene, La Jolla, Calif.).

The nucleotide sequence corresponding to the 3′ end of the cDNA wasdetermined and the plasmid designated Y5-bd-8. However, attempts todetermine the 5′ sequence of the beagle Y5 receptor by 5′ RACE wereunsuccessful.

As a second approach, a canine brain cDNA library (in the pEXJ vector)was screened by PCR using primers BB33 (TM-3) and BB34 (3-4 loop).Vector-anchored PCR, using primers BB34 and KS938 (pEXJ+strand) or KS939(PEXJ − strand) was then used to amplify the 5′ end from two positivepools. The resulting PCR products (0.6 and 0.57 kb) were purified froman agarose gel and subcloned into the pCR Script vector (Stratagene, LaJolla, Calif.). The nucleotide sequence of the longer of these productswas determined using a sequenase kit (USB, Cleveland, Ohio) anddesignated dogY5-16. By comparison to the human Y5 receptor, dogY5-16lacked the first 18 nucleotides of the Y5 coding sequence.

To obtain the additional 5′ sequence, a nitrocellulose membrane(Schleicher and Schuell, Keene, N.H.) containing 20 μg of HindIII-cutcanine genomic DNA (Clontech, Palo Alto, Calif.) was hybridized with a³²P-labeled oligonucleotide probe (BB53) corresponding to nucleotides3-35 of dogY5-16. A 4.2 kb hybridizing band was isolated from areplicate agarose gel and subcloned into the pUC18 vector. Vectoranchored PCR was performed on one-tenth of the ligation reaction usingBB34 (3-4 loop) and BB77 (pUC18 + strand) or BB78 (pUC18 − strand). Theresulting PCR products (1.35, 0.87, 0.75 and 0.7 kb) were thenre-amplified using BB77 and a nested primer BB70 (nucleotides 94-111from dogY5-16). The resulting PCR products (0.4, 0.7 and 0.95 kb) werepurified from an agarose gel and subcloned in pCR Script (Stratagene, LaJolla, Calif.). A portion of the 0.95 kb fragment, designateddogY5-2-29, was sequenced using a Sequenase kit (USB, Cleveland, Ohio).

To obtain a full-length canine Y5 receptor, the primers BB80 (5′untranslated sequence {UT} from dogY5-2-29) and BB54 (carboxy tail and3′ UT from Y5-bd-8) were used to amplify 0.36 μg of beagle genomic DNA.PCR was performed using Expand High Fidelity polymerase (BoehringerMannheim Corporation, Indianapolis, Ind.) under the followingconditions: 94° C. for 1 min, 63° C. for 2 min and 68° C. for 3 min for38 cycles. The resulting 1.4 kb PCR band was purified from an agarosegel and subcloned into pEXJ. Three clones, designated BO10, BO11 andBO12 were sequenced using a sequenase kit (USB, Clevland, Ohio). ThepEXJ derived plasmid comprising clone BO11 was designated cY5-BO11 andwas deposited with the ATCC on May 29, 1996, under ATCC Accession No.97587.

The primers used as described above were as follows:

BB33:

5′-GCCTTTTCTTCAATGTGTGTCAG-3′ (Seq. I.D. No. 15).

BB34:

5′-CCAGACAGTAGCAATCAGGAAGTAGC-3′ (Seq. I.D. No. 16).

KS938:

5′-AAGCTTCTAGAGATCCCTCGACCTC-3′ (Seq. I.D. No. 17).

KS939:

5′-AGGCGCAGAACTGGTAGGTATGGAA-3′ (Seq. I.D. No. 18).

BB53:

5′-GAACTCTAAGATGGATTTAGAACTCCAGATTTT-3′ (Seq. I.D. No. 19).

BB77:

5′-ATGCTTCCGGCTCGTATGTTGTGTGG-3′ (Seq. I.D. No. 20).

BB78:

5′-GCCTCTTCGCTATTACGCCAGCTGGC-3′ (Seq. I.D. No. 21).

BB70:

5′-TAGTCATCCCAGACTGGG-3′ (Seq. I.D. No. 22).

BB80:

5′-GTAGTCTCCCTCTCAGAATTGATTTATCG-3′ (Seq. I.D. No. 23).

BB54:

5′-GGTAAACATGAAGAATTATGACATATGAAGAC-3′ (Seq. I.D. No. 24).

Northern Blots

Human brain multiple tissue northern blots (MTN blots II and III,Clontech, Palo Alto, Calif.) carrying mRNA purified from various humanbrain areas was hybridized at high stringency according to themanufacturer specifications. The probe was a 0.8 kb DNA PCR fragmentcorresponding to the TM III—carboxy end of the 5-6 loop in the codingregion of the human Y5 receptor subtype.

A rat multiple tissue northern blot (rat MTN blot, Clontech, Palo Alto,Calif.) carrying mRNA purified from various rat tissues was hybridizedat high stringency according to the manufacturer specifications. Theprobe was a 0.8 kb DNA PCR fragment corresponding to the TM III—carboxyend of the 5-6 loop in the coding region of the rat Y5 receptor subtype.

Southern Blot

Southern blots (Geno-Blot, clontech, Palo Alto, Calif.) containing humanor rat genomic DNA cut with five different enzymes (8 μg DNA per lane)was hybridized at high stringency according to the manufacturerspecifications. The probe was a 0.8 kb DNA PCR fragment corresponding tothe TM III—carboxy end of the 5-6 loop in the coding region of the humanand rat Y5 receptor subtypes.

Production of Recombinant Baculovirus

A BamHI site directly 5′ to the starting methionine of human Y5 wasgenetically engineered by replacing the beginning ≈100 base pairs of hY5(i.e. from the starting methionine to an internal EcoRI site) with twooverlapping synthetically-derived oligonucleotides (≈100 bases each),containing a 5′ BamHI site and a 3′ EcoRI site. This permitted theisolation of an ≈1.5 kb Bam HI/Hind III fragment containing the codingregion of hY5. This fragment was subcloned into pBlueBacIII™ into theBam HI/Hind III sites found in the polylinker (construct calledpBB/hY5). To generate baculovirus, 0.5 μg of viral DNA (BaculoGold™) and3 μg of pBB/hY5 were co-transfected into 2×10⁶ Spodoptera frugiperdainsect Sf9 cells by calcium phosphate co-precipitation method, asoutlined by Pharmingen (in “Baculovirus Expression Vector System:Procedures and Methods Manual”). The cells were incubated for 5 days at27° C. The supernatant of the co-transfection plate was collected bycentrifugation and the recombinant virus (hY5BB3) was plaque purified.The procedure to infect cells with virus, to prepare stocks of virus andto titer the virus stocks were as described in Pharmingen's manual.

Cell Culture

COS-7 cells were grown on 150 mm plates in D-MEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells were trypsinized and split 1:6 every3-4 days. Human embryonic kidney 293 cells were grown on 150 mm platesin D-MEM with supplements (minimal essential medium) with Hanks' saltsand supplements (Dulbecco's Modified Eagle Medium with 10% bovine calfserum, 4 mM glutamine, 100 units/ml penicillin/100 μg/ml streptomycin)at 37° C., 5% CO₂. Stock plates of 293 cells were trypsinized and split1:6 every 3-4 days. Mouse fibroblast LM(tk-) cells were grown on 150 mmplates in D-MEM with supplements (Dulbecco's Modified Eagle Medium with10% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin) at 37° C., 5% CO₂. Stock plates of LM(tk-) cells weretrypsinized and split 1:10 every 3-4 days.

LM(tk-) cells stably transfected with the human Y5 receptor wereroutinely converted from an adherent monolayer to a viable suspension.Adherent cells were harvested with trypsin at the point of confluence,resuspended in a minimal volume of complete DMEM for a cell count, andfurther diluted to a concentration of 10⁶ cells/ml in suspension media(10% bovine calf serum, 10% 10×Medium 199 (Gibco), 9 mM NaHCO₃, 25 mMglucose, 2 mM L-glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin, and 0.05% methyl cellulose). The cell suspension wasmaintained in a shaking incubator at 37° C., 5% CO₂ for 24 hours.Membranes harvested from cells grown in this manner may be stored aslarge, uniform batches in liquid nitrogen. Alternatively, cells may bereturned to adherent cell culture in complete DMEM by distribution into96-well microtiter plates coated with poly-D-lysine (0.01 mg/ml)followed by incubation at 37° C., 5% CO₂ for 24 hours. Cells prepared inthis manner yielded a robust and reliable NPY-dependent response in cAMPradio-immunoassays as further described hereinbelow.

Mouse embryonic fibroblast NIH-3T3 cells were grown on 150 mm plates inDulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovinecalf serum, 4 mm glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of NIH-3T3 cells weretrypsinized and split 1:15 every 3-4 days.

Sf9 and Sf2 cells were grown in monolayers on 150 mm tissue culturedishes in TMN-FH media supplemented with 10% fetal calf serum, at 27°C., no CO₂. High Five insect cells were grown on 150 mm tissue culturedishes in Ex-Cell 400™ medium supplemented with L-Glutamine, also at 27°C., no CO₂.

Transient Transfection

All receptor subtypes studied (human and rat Y1, human and rat Y2, humanand rat Y4, human, rat and canine Y5) were transiently transfected intoCOS-7 cells by the DEAE-dextran method, using 1 μg of DNA/10⁶ cells(Cullen, 1987). The Y1 receptor was prepared using known methods(Larhammar, et al., 1992).

Stable Transfection

Human Y1, human Y2, and rat Y5 receptors were co-transfected with aG-418 resistant gene into the human embryonic kidney 293 cell line by acalcium phosphate transfection method (Cullen, 1987). Stably transfectedcells were selected with G-418. Human Y4 and human Y5 receptors weresimilarly transfected into mouse fibroblast LM(tk-) cells and NIH-3T3cells. Canine Y5 receptors also may be similarly transfected intoLM(tk-), NIH-3T3 cells or other appropriate host cells. Additional hostcells appropriate for transfection of the Y-type receptors are wellknown in the art and include, but are not limited to, Chinese hamsterovary cells (CHO), the glial cell line C6, or non-mammalian host cellssuch as Xenopus melanophore cells.

Expression of Receptors in Xenopus Oocytes

Expression of genes in Xenopus oocytes is well known in the art(Coleman, Transcription and Translation: A Practical Approach (B. D.Hanes, S. J. Higgins, eds., pp 271-302, IRL Press, Oxford, 1984; Y.Masu, et al. (1987) Nature 329:836-838; Menke, J. G. et al. (1984) J.Biol. Chem. 269(34):21583-21586) and is performed using microinjectioninto Xenopus oocytes of native mRNA or in vitro synthesized mRNA. Thepreparation of in vitro synthesized mRNA can be performed using variousstandard techniques (J. Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Editions, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989) including using T7 polymerase with the mCAP RNAcapping kit (Stratagene).

Expression of other G-protein Coupled Receptors

α₁ Human Adrenergic Receptors: To determine the binding of compounds tohuman α₁ receptors, LM(tk-) cell lines stably transfected with the genesencoding the α_(1a), α_(1b), and α_(1d) receptors were used. Thenomenclature describing the α₁ receptors was changed recently, such thatthe receptor formerly designated α_(1a) is now designated α_(1d), andthe receptor formerly designated α_(1c) is now designated α_(1a) (ref).The cell lines expressing these receptors were deposited with the ATCCbefore the nomenclature change and reflect the subtype designationsformerly assigned to these receptors. Thus, the cell line expressing thereceptor described herein as the α_(1a) receptor was deposited with theATCC on Sep. 25, 1992, under ATCC Accession No. CRL 11140 with thedesignation L-α_(1c). The cell line expressing receptor described hereinas the α_(1d) receptor was deposited with the ATCC on Sep. 25, 1992,under ATCC Accession No. CRL 11138 with the designation L-α_(1A). Thecell line expressing the α_(1b) receptor is designated L-α_(1B), and wasdeposited on Sep. 25, 1992, under ATCC Accession No. CRL 11139.

α₂ Human Adrenergic Receptors: To determine the binding of compounds tohuman α₂ receptors, LM(tk-) cell lines stably transfected with the genesencoding the α_(2A), α_(2B), and α_(2C) receptors were used. The cellline expressing the α_(2A) receptor is designated L-α_(2A), and wasdeposited on Nov. 6, 1992, under ATCC Accession No. CRL 11180. The cellline expressing the α_(2B) receptor is designated L-NGC-α_(2B), and wasdeposited on Oct. 25, 1989, under ATCC Accession No. CRL 10275. The cellline expressing the α_(2C) receptor is designated L-α_(2C), and wasdeposited on Nov. 6, 1992, under ATCC Accession No. CRL-11181. Celllysates were prepared as described below (see Radioligand Binding toMembrane Suspensions), and suspended in 25 mM glycylglycine buffer (pH7.6 at room temperature). Equilibrium competition binding assay wereperformed using [³H]rauwolscine (0.5 nM), and nonspecific binding wasdetermined by incubation with 10 μM phentolamine. The bound radioligandwas separated by filtration through GF/B filters using a cell harvester.

Human Histamine H₁ Receptor: The coding sequence of the human histamineH₁ receptor, homologous to the bovine H₁ receptor, was obtained from ahuman hippocampal cDNA library, and was cloned into the eukaryoticexpression vector pcEXV-3. The plasmid DNA for the H₁ receptor isdesignated pcEXV-H1, and was deposited on Nov. 6, 1992, under ATCCAccession No. 75346. This construct was transfected into COS-7. cells bythe DEAE-dextran method. Cells were harvested after 72 hours and lysedby sonication in 5 mM Tris-HCl, 5 mM EDTA, pH 7.5. The cell lysates werecentrifuged at 1000 rpm for 5 min at 4° C., and the supernatant wascentrifuged at 30,000×g for 20 min. at 4° C. The pellet was suspended in37.8 mM NaHPO₄, 12.2 mM KH₂PO₄, pH 7.5. The binding of the histamine H₁antagonist [³H)mepyramine (1 nM, specific activity: 24.8 Ci/mM) was donein a final volume of 0.25 mL and incubated at room temperature for 60min. Nonspecific binding was determined in the presence of 10 μMmepyramine. The bound radioligand was separated by filtration throughGF/B filters using a cell harvester.

Human Histamine H₂ Receptor: The coding sequence of the human H₂receptor was obtained from a human placenta genomic library, and clonedinto the cloning site of PCEXV-3 eukaryotic expression vector. Theplasmid DNA for the H₂ receptor is designated pcEXV-H2, and wasdeposited on Nov. 6, 1992 under ATCC Accession No. 75345. This constructwas transfected into COS-7 cells by the DEAE-dextran method. Cells wereharvested after 72 hours and lysed by sonication in 5 mM Tris-HCl, 5 mMEDTA, pH 7.5. The cell lysates were centrifuged at 1000 rpm for 5 min at4° C., and the supernatant was centrifuged at 30,000×g for 20 min at 4 °C. The pellet was suspended in 37.8 mM NaHPO₄, 12.2 mM K₂PO₄, pH 7.5.The binding of the histamine H₂ antagonist [³H]tiotidine (5 nM, specificactivity: 70 Ci/mM) was done in a final volume of 0.25 ml and incubatedat room temperature for 60 min. Nonspecific binding was determined inthe presence of 10 μM histamine. The bound radioligand was separated byfiltration through GF/B filters using a cell harvester.

Human serotonin Receptors: 5HT_(1Dα), 5HT_(1Dβ), 5HT_(1E), 5HT_(1F)Receptors: LM(tk-) clonal cell lines stably transfected with the genesencoding each of these 5HT receptor subtypes were prepared as describedabove. The cell line for the 5HT_(1Dα) receptor, designated asLtk-8-30-84, was deposited on Apr. 17, 1990, and accorded ATCC AccessionNo. CRL 10421. The cell for the 5HT_(1Dβ) receptor, designated asLtk-11, was deposited on Apr. 17, 1990, and accorded ATCC Accession No.CRL 10422. The cell line for the 5HT_(1E) receptor, designated 5HT_(1E)-7, was deposited on Nov. 6, 1991, and accorded ATCC AccessionNo. CRL 10913. The cell line for the ⁵HT_(1F) receptor, designatedL-5-HT_(1F), was deposited on Dec. 27, 1991, and accorded ATCC AccessionNo. ATCC 10957. Membrane preparations comprising these receptors wereprepared as described below, and suspended in 50 mM Tris-HCl buffer (pH7.4 at 37° C.) containing 10 mM MgCl₂, 0.2 mM EDTA, 10 μM pargyline, and0.1% ascorbate. The binding of compounds was determined in competitionbinding assays by incubation for 30 minutes at 37° C. in the presence of5 nM [³H]serotonin. Nonspecific binding was determined in the presenceof 10 μM serotonin. The bound radioligand was separated by filtrationthrough GF/B filters using a cell harvester.

Human 5HT₂ Receptor: The coding sequence of the human 5HT₂ receptor wasobtained from a human brain cortex cDNA library, and cloned into thecloning site of pcEXV-3 eukaryotic expression vector. This construct wastransfected into COS-7 cells by the DEAE-dextran method. Cells wereharvested after 72 hours and lysed by sonication in 5 mM Tris-HCl, 5 mMEDTA, pH 7.5. This cell line was deposited with the ATCC on Oct. 31,1989, designated as L-NGC-5HT₂, and was accorded ATCC Accession No. CRL10287. The cell lysates were centrifuged at 1000 rpm for 5 minutes at 4°C., and the supernatant was centrifuged at 30,000×g for 20 minutes at 4°C. The pellet was suspended in 50 mM Tris-HCl buffer (pH 7.7 at roomtemperature) containing 10 mM MgSO₄, 0.5 mM EDTA, and 0.1% ascorbate.The potency of alpha-1 antagonists at 5HT₂ receptors was determined inequilibrium competition binding assays using [3H]ketanserin (1 nM).Nonspecific binding was defined by the addition of 10 μM mianserin. Thebound radioligand was separated by filtration through GF/B filters usinga cell harvester.

Human 5-HT₇ Receptor: A LM(tk-) clonal cell line stably transfected withthe gene encoding the 5HT₇ receptor subtype was prepared as describedabove. The cell line for the 5HT₇ receptor, designated as L-5HT_(4B),was deposited on Oct. 20, 1992, and accorded ATCC Accession No. CRL11166.

Human Dopamine D₃ Receptor: The binding of compounds to the human D3receptor was determined using membrane preparations from COS-7 cellstransfected with the gene encoding the human D₃ receptor. The humandopamine D3 receptor was prepared using known methods. Sokoloff, P. etal., Nature, 347, 146 (1990), and deposited with the European MolecularBiological Laboratory (EMBL) Genbank as X53944). Cells were harvestedafter 72 hours and lysed by sonication in 5 mM Tris-HCl, 5 mM EDTA, pH7.5. The cell lysates were centrifuged at 1000 rpm for 5 minutes at 4°C., and the supernatant was centrifuged at 30,000×g for 20 minutes at 4°C. The pellet was suspended in 50 mM Tris-HCl (pH 7.4) containing 1 mMEDTA, 5 mM KCl, 1.5 mM CaCl₂, 4 mM MgCl₂, and 0.1% ascorbic acid. Thecell lysates were incubated with [³H]spiperone (2 nM), using 10 μM(+)Butaclamol to determine nonspecific binding.

Membrane Harvest

Membranes were harvested from COS-7 cells 48 hours after transienttransfection. Adherent cells were washed twice in ice-cold phosphatebuffered saline (138 mM NaCl, 8.1 mM Na₂HPO₄, 2.5 mM KCl, 1.2 mM KH₂PO₄,0.9 mM CaCl₂, 0.5 mM MgCl₂, pH 7.4) and lysed by sonication in ice-coldsonication buffer (20 mM Tris-HCl, 5 mM EDTA, pH 7.7). Large particlesand debris were cleared by low speed centrifugation (200×g, 5 min, 4°C.). Membranes were collected from the supernatant fraction bycentrifugation (32,000×g, 18 min, 4° C.), washed with ice-cold hypotonicbuffer, and collected again by centrifugation (32,000×g, 18 min, 4° C.).The final membrane pellet was resuspended by sonication into a smallvolume of ice-cold binding buffer (˜1 mL for every 5 plates: 10 mM NaCl,20 mM HEPES, 0.22 mM KH₂PO₄, 1.26 mM CaCl₂, 0.81 mM MgSO₄, pH 7.4).Protein concentration was measured by the Bradford method (Bradford,1976) using Bio-Rad Reagent, with bovine serum albumin as a standard.Membranes were held on ice for up to one hour and used fresh, orflash-frozen and stored in liquid nitrogen.

Membranes were prepared similarly from 293, LM(tk-), and NIH-3T3 cells.To prepare membranes from baculovirus infected cells, 2×10⁷ Sf21 cellswere grown in 150 mm tissue culture dishes and infected with ahigh-titer stock of hY5BB3. Cells were incubated for 2-4 days at 27° C.,no CO₂ before harvesting and membrane preparation as described above.

Membranes were prepared similarly from dissected rat hypothalamus.Frozen hypothalami were homogenized for 20 seconds in ice-coldsonication buffer with the narrow probe of a Virtishear homogenizer at1000 rpm (Virtis, Gardiner, NY). Large particles and debris were clearedby centrifugation (200×g, 5 min, 4° C.) and the supernatant fraction wasreserved on ice. Membranes were further extracted from the pellet byrepeating the homogenization and centrifugation procedure two moretimes. The supernatant fractions were pooled and subjected to high speedcentrifugation (100,000×g, 20 min. 4° C.). The final membrane pellet wasresuspended by gentle homogenization into a small volume of ice-coldbinding buffer (1 mL/gram wet weight tissue) and held on ice for up toone hour, or flash-frozen and stored in liquid nitrogen.

Radioligand Binding to Membrane Suspensions

Membrane suspensions were diluted in binding buffer supplemented with0.1% bovine serum albumin to yield an optimal membrane proteinconcentration so that ¹²⁵I-PYY (or alternative radioligand such as¹²⁵I-NPY, ¹²⁵I-PYY₃₋₃₆, or ¹²⁵I-[Leu³¹Pro³⁴]PYY) bound by membranes inthe assay was less than 10% of ¹²⁵I-PYY (or alternative radioligand)delivered to the sample (100,000 dpm/sample=0.08 nM for competitionbinding assays). ¹²⁵I-PYY (or alternative radioligand) and peptidecompetitors were also diluted to desired concentrations in supplementedbinding buffer. Individual samples were then prepared in 96-wellpolypropylene microtiter plates by mixing ¹²⁵I-PYY (25 μL) (oralternative radioligand), competing peptides or supplemented bindingbuffer (25 μL), and finally, membrane suspensions (200 μl). Samples wereincubated in a 30° C. water bath with constant shaking for 120 min.Incubations were terminated by filtration over Whatman GF/C filters(pre-coated with 1% polyethyleneimine and air-dried before use),followed by washing with 5 mL of ice-cold binding buffer. Filter-trappedmembranes were impregnated with MultiLex solid scintillant (Wallac,Turku, Finland) and counted for ¹²⁵I in a Wallac Beta-Plate Reader.Non-specific binding was defined by 300 nM human NPY for all receptorsexcept the Y4 subtypes; 100 nM human PP was used for the human Y4 and100 nM rat PP for the rat Y4. Specific binding in time course andcompetition studies was typically 80%; most non-specific binding wasassociated with the filter. Binding data were analyzed using nonlinearregression and statistical techniques available in the GraphPAD Prismpackage (San Diego, Calif.).

The canine Y5 receptor pharmacology was investigated using porcine¹²⁵I-PYY as described above. Nonspecific binding was defined by 1 μMhuman NPY. As above, membranes were collected by filtration over WhatmanGF/C filters and counted for radioactivity.

Functional Assay: Radioimmunoassay of cAMP

Stably transfected cells were seeded into 96-well microtiter plates andcultured until confluent. To reduce the potential for receptordesensitization, the serum component of the media was reduced to 1.5%for 4 to 16 hours before the assay. Cells were washed in Hank's bufferedsaline, or HBS (150 mM NaCl, 20 mM HEPES, 1 mM CaCl₂, 5 mM KCl, 1 mMMgCl₂, and 10 mM glucose) supplemented with 0.1% bovine serum albuminplus 5 mM theophylline and pre-equilibrated in the same solution for 20min at 37° C. in 5% CO₂. Cells were then incubated 5 min with 10 μMforskolin and various concentrations of receptor-selective ligands. Theassay was terminated by the removal of HBS and acidification of thecells with 100 mM HCl. Intracellular cAMP was extracted and quantifiedwith a modified version of a magnetic bead-based radioimmunoassay(Advanced Magnetics, Cambridge, Mass.). The final antigen/antibodycomplex was separated from free ¹²⁵I-cAMP by vacuum filtration through aPVDF filter in a microtiter plate (Millipore, Bedford, Mass.). Filterswere punched and counted for ¹²⁵I in a Packard gamma counter. Bindingdata were analyzed using nonlinear regression and statistical techniquesavailable in the GraphPAD Prism package (San Diego, Calif.).

Functional Assay: Intracellular Calcium Mobilization

The intracellular free calcium concentration was measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM(ref). Stably transfected cells were seeded onto a 35 mm culture dishcontaining a glass coverslip insert. Cells were washed with HBS andloaded with 100 μl of Fura-2/AM (10 μM) for 20 to 40 min. After washingwith HBS to remove the Fura-2/AM solution, cells were equilibrated inHBS for 10 to 20 min. Cells were then visualized under the 40×objectiveof a Leitz Fluovert FS microscope and fluorescence emission wasdetermined at 510 nM with excitation wave lengths alternating between340 nM and 380 nM. Raw fluorescence data were converted to calciumconcentrations using standard calcium concentration curves and softwareanalysis techniques.

Tissue Preparation for Neuroanatomical Studies

Male Sprague-Dawley rats (Charles Rivers) were decapitated and thebrains rapidly removed and frozen in isopentane. Coronal sections werecut at 11 μm on a cryostat and thaw-mounted onto poly-L-lysine coatedslides and stored at −80° C. until use. Prior to hybridization, tissueswere fixed in 4% paraformaldehyde, treated with 5 mM dithiothreitol,acetylated in 0.1 M triethanolamine containing 0.25% acetic anhydride,delipidated with chloroform, and dehydrated in graded ethanols.

Probes

The oligonucleotide probes employed to characterize the distribution ofthe rat NPY Y5 mRNA were complementary to nucleotides 1121 to 1165 inthe 5,6-loop of the rat Y5 mRNA (FIG. 3A) 45mer antisense and senseoligonucleotide probes were synthesized on a Millipore Expedite 8909Nucleic Acid Synthesis System. The probes were then lyophilized,reconstituted in sterile water, and purified on a 12% polyacrylamidedenaturing gel. The purified probes were again reconstituted to aconcentration of 100 ng/μL, and stored at −20° C.

In Situ Hybridization

Probes were 3′-end labeled with ³⁵S-dATP (1200 Ci/mmol, New EnglandNuclear, Boston, Mass.) to a specific activity of 10⁹ dpm/μg usingterminal deoxynucleotidyl transferase (Pharmacia). The radiolabeledprobes were purified on Biospin 6 chromatography columns (Bio-Rad;Richmond, Calif.), and diluted in hybridization buffer to aconcentration of 1.5×10⁴ cpm/μL. The hybridization buffer consisted of50% formamide, 4×sodium citrate buffer (1×SSC=0.15 M NaCl and 0.015 Msodium citrate), 1×Denhardt's solution (0.2% polyvinylpyrrolidine, 0.2%Ficoll, 0.2% bovine serum albumin), 50 mM dithiothreitol, 0.5 mg/mlsalmon sperm DNA, 0.5 mg/ml yeast tRNA, and 10% dextran sulfate. Onehundred μL of the diluted radiolabeled probe was applied to eachsection, which was then covered with a Parafilm coverslip. Hybridizationwas carried out overnight in humid chambers at 40 to 55° C. Thefollowing day the sections were washed in two changes of 2×SSC for onehour at room temperature, in 2×SSC for 30 min at 50-60° C., and finallyin 0.1×SSC for 30 min at room temperature. Tissues were dehydrated ingraded ethanols and exposed to Kodak XAR-5 film for 3 days to 3 weeks at−20° C., then dipped in Kodak NTB2 autoradiography emulsion diluted 1:1with 0.2% glycerol water. After exposure at 4° C. for 2 to 8 weeks, theslides were developed in Kodak D-19 developer, fixed, and counterstainedwith cresyl violet.

Hybridization Controls

Controls for probe/hybridization specificity included hybridization withthe radiolabeled sense probe, and the use of transfected cell lines.Briefly, COS-7 cells were transfected (see above) with receptor cDNAsfor the rat Y1, Y2 (disclosed in U.S. Pat. No. 5,545,549, filed Feb. 3,1994), Y4 (disclosed in U.S. Pat. No. 5,516,653, filed Dec. 28, 1993),or Y5. As described above, the transfected cells were treated andhybridized with the radiolabeled Y5 antisense and sense oligonucleotideprobes, washed, and exposed to film for 1-7 days.

Analysis of Hybridization Signals

Sections through the rat brain were analyzed for hybridization signalsin the following manner. “Hybridization signal” as used in the presentcontext indicates the relative number of silver grains observed overneurons in a selected area of the rat brain. Two independent observersrated the intensity of the hybridization signal in a given brain area asnonexistent, low, moderate, or high. These were then converted to asubjective numerical scale as 0, +1, +2, or +3 (see Table 10), andmapped on to schematic diagrams of coronal sections through the ratbrain (see FIG. 11).

Chemical Synthetic Methods

Compounds evaluated in the in vitro Y5 receptor binding and functionalassays, and in vivo feeding assays of the present invention (infra) weresynthesized according to the methods described below. Binding of thecompounds to the human Y1, Y2, Y4 and Y5 receptors was evaluated usingstably transfected 293 or LM(tk-) cells as described above, except thatthe binding data reported for compound 1 at the human Y1 and Y2receptors also included data derived from transiently transfected COS-7cells. Stably transfected cell lines which may be used for bindingexperiments include, for the Y1 receptor, 293-hY1-5 (deposited Jun. 4,1996, under ATCC Accession No. CRL-12121); for the Y2 receptor,293-hY2-10 (deposited Jan. 27, 1994, under ATCC Accession No.CRL-11837); for the Y4 receptor, L-hY4-3 (deposited Jan. 11, 1995, underATCC Accession No. CRL 11779); and for the Y5 receptor, L-hY5-7(deposited Nov. 15, 1995, under ATCC Accession No. CRL 11995).

It is generally preferred that the respective product of each processstep, as described hereinbelow, is separated and/or isolated prior toits use as starting material for subsequent steps. Separation andisolation can be effect by any suitable purification procedure such as,for example, evaporation, crystallization, column chromatography, thinlayer chromatography, distillation, etc. While preferred reactants havebeen identified herein, it is further contemplated that the presentinvention would include chemical equivalents to each reactantspecifically enumerated in this disclosure.

Temperatures are given in degrees Centigrade (° C.). The structure offinal products, intermediates and starting materials is confirmed bystandard analytical methods, e.g., microanalysis and spectroscopiccharacteristics (e.g. MS, IR, NMR). Unless otherwise specified,chromatography is carried out using silica gel. Flash chromatographyrefers to medium pressure column chromatography according to Still etal., J. Org. Chem. 43, 2928 (1978).

Synthesis of Compounds 1, 2, 5, 6, 7, 9, 10, and 11

For Compounds 1, 2, 5, 6, 7, 9, 10, and 11, thin layer chromatographywas performed using the following solvent system:

A1: dichloromethane/methanol  9:1 A2: dichloromethane/methanol 19:1 A3:dichloromethane/methanol/ammonium hydroxide 90:10:1 B1:toluene/ethylacetate  1:1 B2: toluene/ethylacetate 10:1 C1:hexanes/ethylacetate  4:1 C2: hexanes/ethylacetate  3:1 C3:hexanes/ethylacetate  2:1

Compound 1 2,4-Diphenylamino-guinazoline hydrochloride

2-Chloro-4-phenylamino-quinazoline (7.671 g) and aniline (3.627 g) areheated for 3 min to produce a melt which is dissolved in methanol. Theproduct is obtained as its hydrochloride salt upon addition of a slightexcess of 4N HCl in dioxane. Recrystallization from isopropanol yields2,4-diphenylamino-quinazoline hydrochloride, m.p. 319-320° C., FAB-MS(Fast Atom Bombardment Mass Spectroscopy): (M+H)⁺=313. Analytical data:C₂₀H₁₆N₄+HCl+0.5 H₂O, m.p. 319-320° C.

The starting material can be prepared as follows:

a) 2-Chloro-4-phenylamino-quinazoline

A solution of 2,4-dichloro-quinazoline (15 g),N,N-diisopropyl-ethylamine (24.9 ml) and aniline (7.5 ml) in isopropanol(75 ml) is heated to reflux for 45 min. The cold reaction mixture isfiltered and the filtrate is concentrated in vacuo. The residue iscrystallized from diethylether-toluene (1:1) to give2-chloro-4-phenyl-amino-quinazoline, m.p. 194-196° C.

b) 2,4-Dichloro-quinazoline

N,N-Dimethylaniline (114.0 g) is added slowly to a solution of 1H,3H-quinazolin-2,4-dione (146.0 g) in phosphorousoxychloride (535.4 ml)while this mixture is heated up to 140° C. After completion of theaddition reflux is continued for 20 h. The reaction mixture is filteredand evaporated to give a residue which is added to ice and water. Theproduct is extracted with dichloromethane and crystallized fromdiethylether and petroleum diethylether to yield2,4-dichloro-quinazoline, m.p. 115-116° C.

Compound 2 Naphthalene-1-sulfonic acid[6-(4-amino-quinazolin-2-ylamino)-hexyl]-amide

A solution of naphthalene-1-sulfonic acid (6-amino-hexyl)-amide (0.450g) and 2-chloro-quinazolin-4-ylamine (see: U.S. Pat. No. 3,956,495)(0.264 g) in 20 ml of isopentylalcohol is heated up to 120° C. for 15 h.Concentration of the reaction mixture followed by chromatography onsilica gel (B1) yields naphthalene-1-sulfonic acid[6-(4-amino-quinazolin-2-ylamino)-hexyl]-amide as a white powder,melting at 98-101° C. Rf(B1) 0.28, FAB-MS: (M+H)⁺=450. AnalyticalC₂₆H₂₉N₅O₂S+HCl+H₂O+0.6 1,4 dioxane. m.p. 98-101° C.

Compound 5 trans-Naphthalene-1-sulfonic acid{4-[(4-amino-quinazolin-2-ylamino)-methyl]-cyclohexylmethyl}-amidehydrochloride

A suspension of 2-chloro-quinazolin-4-ylamine (7.02 g) andtrans-naphthalene-1-sulfonic acid (4-aminomethyl-cyclohexylmethyl)-amide(13 g) in 250 ml of isopentyl-alcohol is heated up to 120° C. for 15 h.The resulting solution is concentrated and chromatographed (silica gel,B2) to give the product as a foam. This material is taken up indichloromethane (250 ml) and treated at 0° C. with a 4 N HCl solution indioxane (10 ml). Concentration in vacuo provides a foam which istriturated in boiling cyclohexane to yield after filtrationtrans-naphthalene-1-sulfonic acid{4-[(4-amino-quinazolin-2-ylamino)-methyl]-cyclohexylmethyl}-amidehydrochloride melting at 155-164° C. Rf(B2) 0.23, FAB-MS: (M+H)⁺=476.m.p. 155-164° C.

The starting material is prepared as follows:

a) trans-(4-Hydroxymethyl-cyclohexylmethyl)-carbamic acid tert-butylester

A solution oftrans-4-(tert-butoxycarbonylamino-methyl)-cyclohexanecarboxylic acid(obtained according to: EP 0614 911 A1) (34.5 g) and triethylamine (28ml) in dichloromethane (700 ml) is cooled to −70° C. and treated withmethylchloroformate (12.9 ml). The reaction mixture is stirred 0.5 h at−70° C. The temperature is allowed to increase to 0° C. and the solutionis stirred another 0.5 h until completion of the reaction. The reactionmixture is taken up in ice-cold dichloromethane, washed with an ice-cold0.5 N HCl solution, a saturated aqueous sodium carbonate solution andwater. The organics are dried over sodium sulfate and concentrated to41.3 g of mixt-anhydride as an oil. This material is taken up in THF andtreated at −70° C. with sodium borohydride (5.90 g), followed byabsolute methanol (10 ml). The reaction mixture is stirred 15 h at 0° C.and 1 h at ambient temperature to drive the reaction to completion. A0.5N HCl solution is then carefully added at 0° C., followed by ethylacetate. The organics are washed with a saturated aqueous sodiumcarbonate solution, water, dried over sodium sulfate and concentrated.Chromatography on silica gel (Al) yieldstrans-(4-hydroxymethyl-cyclohexylmethyl)-carbamic acid tert-butyl esteras a white powder, melting at 88-89° C. Rf(A1) 0.24.

b) trans-(4-Azidomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester

trans-(4-Hydroxymethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester(24 g) in pyridine (200 ml) at 0° C. is treated with a solution ofpara-toluenesulfonylchloride (24.44 g) in pyridine (50 ml). The reactionmixture is stirred at 0° C. until completion and concentrated in vacuo.The residue is taken up in ethyl acetate, washed with water and driedover sodium sulfate. Concentration of the solution yields the tosylate,used without further purification. This material is treated with sodiumazide (19.23 g) in N,N-dimethylformamide (800 ml) at 50° C. Aftercompletion of the reaction, the solution is concentrated and theresulting paste is taken up in dichloromethane, washed with water andconcentrated. Chromatography of the crude material on silica gel (A2then A3) provides trans-(4-azidomethyl-cyclohexylmethyl)-carbamic acidtert-butyl ester as an oil. Rf(A3) 0.33; IR (dichloromethane) λ max 2099cm⁻¹.

c) trans-(4-Aminomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester

trans-(4-Azidomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester(24 g) in ethyl acetate (1 liter) is hydrogenated over platinumoxide(2.4 g) at ambient temperature under atmospheric pressure of hydrogen.The catalyst is filtered-off and the filtrate concentrated to yieldtrans-(4-aminomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester asan oil. Rf(C2) 0.41.

d)trans-(4-[(Naphthalene-1-sulfonylamino)-methyl]-cyclohexylmethyl)-carbamicacid tert-butyl ester

A solution of trans- (4-aminomethyl-cyclohexylmethyl)-carbamic acidtert-butyl ester (17 g) and ethyldiisopropylamine (14.41 ml) inN,N-dimethylformamide (350 ml) is cooled to 0° C. and treated with asolution of naphthalene-1-sulfonylchloride (15.9 g) inN,N-dimethylformamide (100 ml). The reaction is stirred at ambienttemperature for 2 h, concentrated in vacuo. The residue is taken up indichloromethane, washed with a 0.5 N HCl solution, a saturated aqueoussodium carbonate solution and water, dried and concentrated.Crystallization from hexanes-ethyl acetate gives trans-(4-[(naphthalene-1-sulfonylamino)-methyl]-cyclohexylmethyl)-carbamic acidtert-butyl ester as a white powder, melting at 199-200° C. Rf(A1) 0.42.

e) trans-Naphthalene-1-sulfonic acid(4-aminomethyl-cyclohexylmethyl)-amide

A suspension oftrans-{4-[(naphthalene-1-sulfonylamino)-methyl]-cyclohexylmethyl}-carbamicacid tert-butyl ester (25 g) in chloroform (300 ml) is treated with a 4N HCl solution in dioxane (300 ml) at 0° C. After completion, thereaction mixture is concentrated in vacuo, the residue is taken up in a1 N sodium hydroxide solution and dichloromethane. After extraction withdichloromethane, the organics are dried over sodium sulfate andconcentrated to 18.5 g of trans-naphthalene-1-sulfonic acid(4-aminomethyl-cyclohexylmethyl) -amide as a white powder melting at157-162° C. Rf(C3) 0.36.

Compound 6 2-[4-(Piperidin-1-yl)-phenylamino]-4-Phenylamino-quinazolinedihydrochloride

A mixture of 2-chloro-4-phenylamino-quinazoline (0.18 g) andN-(4-aminophenyl)-piperidine (0.164 g) is heated for 3 min to produce amelt which is dissolved in isopropanol (4 ml). 4 N HCl in dioxane (1 ml)is added. Recrystallization from ethanol and diethylether yields2-[4-(piperidin-1-yl)-phenylamino]-4-phenylamino-quinazolinedihydrochloride, Rf (A1) 0.64, FAB-MS: (M+H)⁺=396. m.p.:(decomposition).

Compound 7 trans-2-(4-Acetoxy-cyclohexylamino)-4-Phenylamino-quinazolinehydrochloride

A solution oftrans-2-(4-hydroxy-cyclohexyamino)-4-phenylamino-quinazolinehydrochloride (1.3 g) and acetic anhydride (0.33 ml) in acetic acid (5ml) is stirred at ambient temperature for 16 h. The solvent is removedin vacuo and the residue is added to 2N aqueous NaOH. Extraction withethyl acetate followed by chromatography on silica gel (A4) gives acrude product which is treated with 4 N HCl in dioxane. Crystallizationfrom acetonitrile and acetone yieldstrans-2-(4-acetoxy-cyclohexylamino)-4-phenylamino-quinazolinehydrochloride, m.p. 217-220° C.; FAB-MS: (M+H)⁺=377; analytical data:C₂₂H₂₄N₄O₂+HCI.

The starting material is prepared as follows:

a) 2-(4-Hydroxy-cyclohexyamino)-4-phenylamino-quinazoline hydrochloride

A mixture of 2-chloro-4-phenylamino-quinazoline (2.3 g) andtrans-4-amino-cyclohexanol (1.26 g) is heated for 3 min to produce amelt which is dissolved in isopropanol. 4 N HCl in dioxane (0.1 ml) isadded. Crystallization from isopropanol and acetone yields2-(-4-hydroxy-cyclohexyamino)-4-phenylamino-quinazoline hydrochloride,m.p. 258-259° C.

Compound 9 8-Methoxy-2-(4-methoxy-phenylamino)-4-phenylamino-quinazolinehydrochloride

A mixture of 2-chloro-8-methoxy-4-phenylamino-quinazoline (1.20 g) and4-methoxy-aniline (0.66 g) is heated for 3 min to produce a melt whichis dissolved in isopropanol (15 ml). 4N HCl in dioxane (0.2 ml) isadded. Crystallization from isopropanol and diethylether yields8-methoxy-2-(4-methoxy-phenylamino)-4-phenylamino-quinazolinedihydrochloride, m.p. 287-289° C., FAB-MS: (M+H)⁺=373. Analytical data:C₂₂H₂₀N₄O₂+HCl.

The starting material can be prepared as follows:

a) 2-Chloro-8-methoxy-4-phenylamino-quinazoline

A solution of 2,4-dichloro-8-methoxy-quinazoline (prepared as describedin J. Chem. Soc. 1948, 1759) (0.6 g), N,N-diisopropyl-ethylamine (0.87ml), and aniline (0.26 ml) in isopropanol (10 ml) is heated to refluxfor 45 min. The cold reaction mixture is filtered and residue iscrystallized from dichloromethane and hexanes to give2-chloro-8-methoxy-4-phenylamino-quinazoline, m.p. 245-246° C.

Compound 10N-Methyl-[4-(6-methoxy-4-phenylamino-quinazolin-2-ylamino)-phenyl1-methanesulfonamidehydrochloride

A solution of 2-chloro-6-methoxy-4-phenylamino-quinazoline (1.15 g) andN-methyl-(4-aminophenyl)-methanesulfonamide (prepared as described inTetrahedron Letters 1992, 33, 8011) (0.89 g) in 5 mL of isopentylalcoholis stirred under nitrogen at 180° C. for 20 min in a sealed vessel. Thewarm reaction mixture is diluted with methanol and the hydrochloridesalt, which is crystallizing on cooling, is filtered off. The crudeproduct is redissolved in ethylacetate and aqueous sodium carbonatesolution and extracted with ethylacetate. The organic extracts are driedand evaporated and the solid residue is titurated with diethylether togiveN-methyl-[4-(6-methoxy-4-phenylamino-quinazolin-2-ylamino)-phenyl]-methanesulfonamideas light yellow crystals melting at 212-215° C.; (Rf (A2) 0.16.Recrystallisation from methanolic hydrogen chloride and diethyletheryieldsN-methyl-[4-(6-methoxy-4-phenylamino-quinazolin-2-ylamino)-phenyl]-methanesulfonamidehydrochloride as light yellow crystals melting at 264-268° C.; Rf (A2)0.16, FAB-MS: (M+H)⁺=450. Analytical data: C₂₃H₂₃N₅O₃S+HCl.

The starting material can be prepared as follows:

a) 2-Chloro-6-methoxy-4-phenylamino-quinazoline

In a procedure analogous to that of Example 1a2,4-dichloro-6-methoxy-quinazoline (1.53 g) (prepared as described in J.Chem. Soc. 1948, 1759), aniline (0.8 g) (0.184 g) andN,N-diisopropyl-ethylamine (1.72 g) are reacted together to give2-chloro-6-methoxy-4-phenylamino-quinazoline as light yellow crystalsmelting at 177-179° C., Rf (A2) 0.59.

Compound 11N-Methyl-[4-(4-Phenylamino-quinazolin-2-ylamino)-phenyl]-methanesulfonamidehydrochloride

A solution of 2-chloro-4-phenylamino-quinazoline (0.92 g) (prepared asdescribed in Example 1a and N-methyl-(4-aminophenyl)-methanesulfonamide(0.80 g) in 10 ml of isopentylalcohol is stirred under nitrogen at 170°C. for 15 min in a sealed vessel. The warm reaction mixture is dilutedwith 10 ml ethanol and the hydrochloride salt, which is crystallizing oncooling, is filtered off to yieldN-methyl-[4-(4-phenylamino-quinazolin-2-ylamino)-phenyl]-methanesulfonamidehydrochloride as light yellow crystals melting at 259-263° C.; Rf (A2)0.11, FAB-MS: (M+H)⁺=420. Analytical data: C₂₂H₂₁N₅O₂S+HCl.

Synthesis of Compounds 17-23, Compound 26 and Compound 27

Compounds 17-23, 26 and 27 were synthesized according to the generalmethod in Scheme 1, as described below. An example of the synthesis of aspecific compound, Compound 17, follows the general description.Compounds 18-23, 26 and 27 were synthesized in the same manner but usingthe appropriately substituted starting materials.

Preparation of the compounds of the present invention having thestructure shown in Formula 1-3, Scheme 1, is carried out usingwell-known methodology for the preparation of a sulfonamide from anamine. Preferably the appropriate arylsulfonyl halide, preferably thechloride (i.e., Ar—SO₂Cl), is reacted with a monoprotected linear orcyclic alkylamine (Krapcho and Kuell, Synth. Comm. 20(16):2559-2564,1990) comprising H₂N-L-K″, where K″ comprises methylene, in the presenceof a base such as a tertiary amine, e.g., triethylamine,dimethylaminopyridine, pyridine or the like, in an appropriate solvent(e.g. CHCl₃, CH₂Cl₂) as shown in Scheme 1, step A, followed bydeprotection of the resulting amine as shown in Scheme 1, Step B, allunder mild conditions (typically room temperature), to yield thedeprotected amine of Formula 1-1. The arylsulfonyl halides are eitherknown in the art or can be prepared according to methods well known inthe art.

Compounds of Formula 1-2 in Scheme 1, may be synthesized from thecompound-of Formula 1-1 by amidation using suitable methods such asthose taught in “The Peptides,” Vol. 1 (Gross and Meinehofer, Eds.Acaemic Press, N.Y., 1979). For example, the compound of Formula 1-1 maybe treated with a carboxylic acid derivative of W in the presence of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) anddimethylaminopyridine (DMAP) in a suitable solvent such as CH₂Cl₂ asshown in Scheme 1, Step C, at room temperature in an inert atmosphere ofargon or nitrogen, to yield the amide compound of Formula 1-2. The K″amine and the carboxylic acid carbon attached to W together form K inthe product.

Alternatively, the compound of Formula 1-2 may be synthesized byacylation of the amine of Formula 1-1 using the acid chloride of W,i.e., WCOCl, in a solvent such as CH₂Cl₂ and a suitable tertiary aminesuch as triethylamine, at room temperature. Again, the K″ amine and theacid chloride carbon attached to W together form K in the product.

The product compounds of Formula 1-3 are then formed by reduction of theamide of Formula 1-3 using borane-tetrahydorfuran (THF) complex, in THFas shown in Scheme 1, Step D, at elevated temperature in an inertatmosphere.

As a specific example of the synthesis of compounds 17-23, 26 and 27,the synthesis of Compound 17 is given hereinbelow.

Compound 17 Naphthalene-2-sulfonic acid (4-[{(1, 2, 3,4-tetrahydronaphthalen-2-yl)methyl}-amino]-trans-cyclohexylmethyl)-amide Step A Scheme 1{4-[(Naphthalene-2-sulfonylamino)-trans-cyclohexylmethyl]-carbamic acidtert-butyl ester

To a stirred solution of (4-aminomethyl-cyclohexylmethyl)carbamic acidtert-butyl ester (0.50 g, 2.1 mmol) and triethyl amine (0.42 g, 4.2mmol) in 50 mL methylene chloride was added 2-naphthalenesulfonylchloride (0.51g, 2.3 mmol). The reaction mixture was stirred for 6 h atroom temperature, quenched with brine, and extracted with methylenechloride (2×50 mL). The organic layer was washed with brine, dried overanhydrous sodium sulfate, and concentrated in vacuo to yield the titledcompound as white solid (0.74 g, 83%): mp 114-5° C.

Step B, Scheme 1 Naphthalene-2-sulfonicacid-(4-aminomethyl-trans-cyclohexylmethyl)-amide

To a stirred solution of (4-[(naphthalene-2-sulfonylamino)-transcyclohexylmethyl]-carbamic acid tert-butyl ester (0.73 g, 1.6 mmol) in25 mL of methylene chloride at room temperature was added 3 mL ofsaturated HCl solution in ethyl acetate and stirred for 4 h. Theprecipitated solid was filtered to yield the titled compound as whitesolid (0.58 g, 99%); mp 286-7° C.

Step C, Scheme 1 1, 2, 3, 4-Tetrahydronaphthalene-2-carboxylicacid[4{(naphthalen-2-sulfonylamino)methyl}-tans-cyclohexylmethyl]amide

A mixture of naphthalene-2-sulfonicacid-(4-aminomethyl-trans-cyclohexylmethyl)amide (0.5 g, 1.4 mmol), EDC(0.54 g, 2.8 mmol), and DMAP (0.34 g, 2.8 mmol) in methylene chloride(30mL) was stirred at room temperature for 0.5 h.1,2,3,4-tetrahydronaphthalen-2-carboxylic acid (0.24 g, 1.4 mmol) wasadded to the reaction mixture and stirred at room temperature till thecompletion of the reaction (by TLC). The reaction mixture was washedwith saturated ammonium chloride (3×30 mL), dried over sodium sulfateand concentrated in vacuo. The residue was flash chromatographed oversilica gel to afford white solid (0.66 g, 99%); mp 225-6° C.

Step D, Scheme 1 Naphthalene-2-sulfonic acid(4-[{(1, 2, 3,4-tetrahydronaphthalen-2-yl)methyl}-amino]-trans-cyclohexylmethyl)-amide

To a solution of 1, 2, 3, 4-tetrahydronaphthalen-2-carboxylicacid[4-{(naphthalen-2-sulfonylamino)methyl}-tanscyclohexylmethyl]amide(0.65g, 1.3 mmol) in tetrahydrofuran (5 mL) cooled to 0° C. was added 6.6 mL1M solution of borane:THF complex and the reaction mixture was refluxedfor 12 h. The reaction mixture was cooled in ice bath and quenched with2 mL of 1N HCl. The reaction mixture was neutralized with 10% aqueoussodium hydroxide solution and extracted with ethyl acetate (3×25 mL).Organic phase was washed with the brine, dried over sodium sulfate,evaporated in vacuo to afford an oil which was purified by preparativeTLC to afford the titled compound(0.44. g, 70%); hydrochloride salt mp(210° C.).

In order to synthesize compounds 18-24, 26 and 27, the2-naphthalenesulfonyl chloride of Step A above, which comprises the “Ar”moiety of Table 2, is replaced with the appropriate Ar-sulfonylchloride, and the 1,2,3,4-tetrahydronaphthalen-2-carboxylic acid used inStep C above, which comprises the “W” moiety of Table 2, is replacedwith the appropriate W-carboxylic acid, to yield product containing thecorresponding Ar and W moieties shown in Table 2.

Synthesis of Compound 25

Compound 25 was synthesized according to Scheme 2. After protection ofH₂N-L-COOH with Boc anhydride in CH₂Cl₂, as shown in Scheme 2, Step A,the protected amine may be amidated with W-K′″ as in Scheme 2, Step B,where K′″ is (CH₂)_(j)CHR₇—NH₂, where R₇ is an ester and j is 1 usingEDC and DMAP in a suitable solvent such as CH₂Cl₂, to yield compounds ofFormula 3-1, where K′″ and the carboxylic acid carbonyl of H₂N-L-COOHtogether form K. The compounds of Formula 3-1 may be deprotected usingwell known methods as shown in Scheme 2, Step C, and furthersulfonylated with a sulfonyl halide of Ar, as shown in Scheme 2, Step D,in a suitable solvent such as CH₂Cl₂ and a tertiary amine such astriethylamine, to form the compound of Formula 3-3. Compounds of Formula3-3 may be reduced to yield the compounds of Formula 3-3, as shown inScheme 2, Step E, using borane-tetrahydorfuran (THF) complex, in THF, atelevated temperature in an inert atmosphere.

A detailed description of the synthesis of Compound 25 is given below:

Compound 25trans-3-(4-Chloro-phenyl)-2-({[4-(naphthalene-1-sulfonylamino)-methyl]-cyclohexanecarbonyl}-amino]-propionicacid methyl ester (a) Step A, Scheme 2trans-4-(tert-Butoxycarbonylamino-methyl)-cyclohexanecarboxylic acid

To a solution of trans-4-(aminomethyl)cyclohexanecarboxylic acid (10 g,57 mmol) in 1 N NaOH (110 mL) cooled to 0° C. was added a solution ofdi-tert-butyl dicarbonate (15 g, 69 mmol) in dioxane (50 mL). Thereaction mixture was stirred at 0° C. for 12 h. The reaction mixture wasneutralized by 1 N HCl solution to pH 3, extracted with ethyl ether(2×300 mL), washed with brine (2×300 mL), dried over anhydrous magnesiumsulfate, and concentrated in vacuo to afford the titled compound (16 g,100%); white solid, mp 128-9° C.

(b) Step B, Scheme 2trans-2-{[4-(tert-Butoxycarbonylamino-methyl)-cyclohexanecarbonyl]-amino}3-(4-Chloro-phenyl)-propionicacid methyl ester

Using the general procedure described for the preparation Step B, Scheme2, trans-4-(tert-butoxycarbonylamino-methyl)-cyclohexanecarboxylic acid(1.1 g, 4.0 mmol) was acylated with D,L-4-chlorophenylalanine methylester hydrochloride (1.0 g, 4.0 mmol) to afford the titled compound (1.9g, 99%); white solid, mp 178-9° C.

(c) Step C, Scheme 2 trans-2-[4-(Aminomethyl-cyclohexanecarbonyl)-amino]3-(4-chloro-phenyl)-propionic acid methyl ester hydrochloride

Using the general procedure described in step C Scheme 2,trans-2-([4-(tert-butoxycarbonylamino-methyl)-cyclohexanecarbonyl]-amino}3-(4-chloro-phenyl)-propionic acid methyl ester (1.8 g, 4.3 mmol) wasdeprotected using HCl in ethyl acetate to afford the titled compound;light yellow solid mp 146-9° C.

(d) Step D, Scheme 2trans-3-(4-Chloro-phenyl)-2-(([4-(naphthalene-1-sulfonylamino)-methyl]-cyclohexanecarbonyl)-amino]-propionicacid methyl ester

Using the general procedure described in step B Scheme 2,trans-2-[4-(aminomethyl-cyclohexanecarbonyl)-amino]3-(4-Chloro-phenyl)-propionic acid methyl ester hydrochloride (0.35 g,0.86 mmol) was sulfonylated with 1-naphthalenesulfonyl chloride (0.42 g,91%) to afford the titled compound; white solid, mp 84-6° C.

Compound 25 was synthesized from the above compound by borane-THFreduction as follows:

(e) step E, Scheme 2 Naphthalene-1-sulfonic Acidtrans-(4-{[2-(4-Chlorophenyl)-1-hydroxymethyl-ethylamino]-methyl}-cyclohexylmethyl)-amide

Using the general procedure described in Step E, Scheme 2,trans-3-(4-chloro-phenyl)-2-({[4-(naphthalene-1-sulfonylamino)-methyl]-cyclohexanecarbonyl}-amino]-propionicacid methyl ester (0.30 g, 0.55 mmol) was reduced by borane:THF complex(1.0 M in THF) to afford the titled compound; colorless oil.

Synthesis of Compound 28 2-(Naphthalen-1-ylamino)-3-phenylpropionitrile

To a solution of 1-naphthalenemethylamine (2.9 g, 20 mmol) andbenzylaldehyde (2.0 g, 17 mmol) in 30 ml of CHCl₃ and 10 ml of MeOH wasadded TMSCN (6.6 ml, 51 mmol) and the resulting solution was stirred for12 h at 25° C. The reaction mixture was concentrated in vacuo, yieldingan oil which was subjected to column chromatography (EtOAc, neat) toprovide 3.5 g (74%) of the desired product as a colorless oil. Productwas identified by NMR.

2-(Naphthalen-1-yl)-3-phenylpropane-1,2-diamine

To a solution of the nitrile (0.5 g, 1.8 mmol) in THF was added 6.9 mlof 1N LiAlH₄ in THF dropwise and the resulting solution was stirred for2 h. The reaction was quenched by adding a few pieces of ice into thesolution. The reaction mixture was diluted with EtOAc and filteredthrough pad of Celite. Organic filtrate was concentrated in vacuo toprovide a oily residue which was subjected to column chromatography(EtOAc, neat) to provide 0.28 g (57%) of the desired product as acolorless oil. The product was identified by NMR.

TABLE 2 No. Ar X R₁ L K W mp Analysis 17

— H

CH₂NHCH₂

210 C₂₉H₃₆N₂O₂S + HCl 19

— H

CH₂NHCH₂

220 C₂₉H₃₆N₂O₂S + HCl + 0.15 CH₂Cl₂ 20

— H

CH₂NHCH₂

200-2 C₂₅H₃₃N₃O₄S + HCl 21

— H

CH₂NHCH₂

171-4 C₂₆H₂₉N₂O₂SF₃ + HCl + 0.075 CHCl₃ 22

— H

CH₂NHCH₂

175-7 C₂₅H₃₅N₃O₂S + 2 HCl + 0.8 Et₂O 23

— H

CH₂NHCH₂

216-7 C₂₆H₂₉N₂O₂SF₃ + HCl 25

— H

223-3 C₂₇H₃₃N₂O₃SCl + HCl 26

— H

CH₂NHCH₂

89 dec C₂₄H₂₈N₄O₄S + 2 HCl 27

— H

CH₂NHCH₂

104-6 C₂₅H₂₈N₄O₄S + 2 HCl + 0.2 CHCl₃

In vivo Studies in Rats Food Intake in Satiated Rats

For these determinations food intake may be measured in normal satiatedrats after intracerebroventricular application (i.c.v.) of NPY in thepresence or absence of the test compound. Male Sprague Dawley rats(Ciba-Geigy AG, Sisseln, Switzerland) weighing between 180 g and 220 gare used for all experiments. The rats are individually housed instainless steel cages and maintained on an 11:13 h light-dark cycle(lights off at 18:00 h) at a controlled temperature of 21-23° C. at alltimes. Water and food (NAFAG lab chow pellets NAFAG, Gossau,Switzerland) are available ad libidum.

Rats under pentobarbital anesthesia are stereotaxically implanted with astainless steel guide cannula targeted at the right lateral ventricle.Stereotaxic coordinates, with the incisor bar set −2.0 mm belowinteraural line, are: −0.8 mm anterior and +1.3 mm lateral to bregma.The guide cannula is placed on the dura. Injection cannulas extend theguide cannulas −3.8 mm ventrally to the skull surface. Animals areallowed at least 4 days of recovery postoperatively before being used inthe experiments. Cannula placement is checked postoperatively by testingall rats for their drinking response to a 50. ng intracerebroventricular(i.c.v.) injection of angiotensin II. Only rats which drink at least 2.5ml of water within 30 min. after angiotensin II injection are used inthe feeding studies.

All injections are made in the morning 2 hours after light onset.Peptides are injected in artificial cerebrospinal fluid (ACSF) in avolume of 5 μl. ACSF contains: NaCl 124 mM, KCl 3.75 mM, CaCl₂ 2.5 mM,MgSO₄ 2.0 mM, KH₂PO₄ 0.22 mM, NaHCO₃ 26 mM and glucose 10 mM.Porcine-NPY (p-NPY) are dissolved in artificial cerebrospinal fluid(ACS). For i.c.v. injection the test compounds are preferably dissolvedin DMSO/water (10%, v/v). The vehicle used for intraperitoneal (i.p.),subcutaneous (s.c.) or oral (p.o.) delivery of compounds is preferablywater, physiological saline or DMSO/water (10% v/v), or cremophor/water(20% v/v), respectively.

Animals which are treated with both test compounds and porcine-NPY aretreated first with the test compound. Then, 10 min. after i.c.v.application of the test compound or vehicle (control), or for i.p.,s.c., or p.o. administration, 30-60 min after application of the testcompound or vehicle, generally, NPY is administered byintracerebroventricular (i.c.v.) application.

Food intake may be measured by placing preweighed pellets into the cagesat the time of NPY injection. Pellets are then removed from the cagesubsequently at each selected time point and replaced with a new set ofpreweighed pellets. The food intake of animals treated with testcompound may be calculated as a percentage of the food intake of controlanimals i.e., animals treated with vehicle. Alternatively, food intakefor each group of animals subjected to a particular experimentalcondition may be expressed as the mean±S.E.M. Statistical analysis isperformed by analysis of variance using the Student-Newman-Keuls test.

Food Intake in Food-deprived Rats

Food-deprivation experiments are conducted with male Sprague-Dawley ratsweighing between 220 g and 250 g. After receipt, the animals areindividually housed for the duration of the study and allowed freeaccess to normal food together with tap water. The animals aremaintained in a room with a 12 h light/dark cycle (8:00 a.m. to 8:00p.m. light) at 24° C. and monitored humidity. After placement intoindividual cages the rats undergo a 4 day equilibration period, duringwhich they are habituated to their new environment and to eating apowdered or pellet diet NAFAG, Gossau, Switzerland).

At the end of the equilibration period, food is removed from the animalsfor 24 hours starting at 8:00 a.m. At the end of the fasting periodcompound or vehicle may be administered to the animals orally or byinjection intraperitoneally or intravenously. After 10-60 min. food isreturned to the animals and their food intake is monitored at varioustime periods during the following 24 hour period. The food intake ofanimals treated with test compound may be calculated as a percentage ofthe food intake of control animals (i.e., animals treated with vehicle).Alternatively, food intake for each group of animals subjected to aparticular experimental condition may be expressed as the mean±S.E.M.

Food Intake in Obese Zucker Rats

The antiobesity efficacy of the compounds according to the presentinvention might also be manifested in Zucker obese rats, which are knownin the art as an animal model of obesity. These studies are conductedwith male Zucker fatty rats (fa/fa Harlan CPB, Austerlitz NL) weighingbetween 480 g and 500 g. Animals are individually housed in metabolismcages for the duration of the study and allowed free access to normalpowdered food and water. The animals are maintained in a room with a 12h light/dark cycle (light from 8:00 A.M. to 8:00 P.M.) at 24° C. andmonitored humidity. After placement into the metabolism cages the ratsundergo a 6 day equilibration period, during which they are habituatedto their new environment and to eating a powdered diet. At the end ofthe equilibration period, food intake during the light and dark phasesis determined. After a 3 day control period, the animals are treatedwith test compounds or vehicle (preferably water or physiological salineor DMSO/water (10%, v/v) or cremophor/water (20%, v/v)). Food intake isthen monitored over the following 3 day period to determine the effectof administration of test compound or vehicle alone. As in the studiesdescribed hereinabove, food intake in the presence of drug may beexpressed as a percentage of the food intake of animals treated withvehicle, or as the amount of food intake for a group of animalssubjected to a particular experimental condition.

Materials

Cell culture media and supplements are from Specialty Media (Lavallette,N.J.). Celliculture plates (150 mm and 96-well microtiter) were fromCorning (Corning, N.Y.). Sf9, Sf21, and High Five insect cells, as wellas the baculovirus transfer plasmid, pBlueBacIII™, were purchased fromInvitrogen (San Diego, Calif.). TMN-FH insect medium complemented with10% fetal calf serum, and the baculovirus DNA, BaculoGold™, was obtainedfrom Pharmingen (San Diego, Calif.). Ex-Cell ₄₀₀™ medium withL-Glutamine was purchased from JRH Scientific. Polypropylene 96-wellmicrotiter plates were from Co-star (Cambridge, Mass.). All radioligandswere from New England Nuclear (Boston, Mass.). Commercially availableNPY and related peptide analogs were either from Bachem California(Torrance, Calif.) or Peninsula (Belmont, Calif.); [D-Trp³²]NPY and PPC-terminal fragments were synthesized by custom order from ChironMimotopes Peptide Systems (San Diego, Calif.). Bio-Rad Reagent was fromBio-Rad (Hercules, Calif.). Bovine serum albumin (ultra-fat free,A-7511) was from Sigma (St. Louis. Mo.). All other materials werereagent grade.

Experimental Results cDNA Cloning

In order to clone a rat hypothalamic “atypical” NPY receptor subtype,applicants used an expression cloning strategy in COS-7 cells (Gearinget al, 1989; Kluxen et al, 1992; Kiefer et al, 1992). This strategy waschosen for its extreme sensitivity since it allows detection of a single“receptor positive” cell by direct microscopic autoradiography. Sincethe “atypical” receptor has only been described in feeding behaviorstudies involving injection of NPY and NPY related ligands in rathypothalamus (see introduction), applicants first examined its bindingprofile by running competitive displacement studies of ¹²⁵I-PYY and¹²⁵I-PYY₃₋₃₆ on membranes prepared from rat hypothalamus. Thecompetitive displacement data indicate: 1) Human PP is able to displace20% of the bound ¹²⁵I-PYY with an IC₅₀ of 11 nM (FIG. 1 and Table 3). Ascan be seen in Table 5, this value does not fit with the isolated ratY1, Y2 and Y4 clones and could therefore correspond to another NPY/PYYreceptor subtype. 2) [Leu₃₁, Pro₃₄] NPY (a Y1 specific ligand) is ableto displace with high affinity (IC₅₀ of 0.38) 27% of the bound²⁵I-PYY₃₋₃₆ ligand (a Y2 specific ligand) (FIG. 2 and Table 3). Thesedata provide the first evidence based on a binding assay that rathypothalamic membranes could carry an NPY receptor subtype with a mixedY1/Y2 pharmacology (referred to as the “atypical” subtype) which fitswith the pharmacology defined in feeding behavior studies.

TABLE 3 Pharmacological Profile of the Rat Hypothalamus

Binding data reflect competitive displacement of ¹²⁵I-PYY and¹²⁵I-PYY₃₋₃₆ from rat hypothalamic membranes. Peptides were tested atconcentrations ranging from 0.001 nM to 100 nM unless noted. The IC₅₀value corresponding to 50% displacement, and the percentage ofdisplacement relative to that produced by 300 nM human NPY, weredetermined by nonlinear regression analysis. Data shown arerepresentative of at least two independent experiments.

TABLE 3 IC₅₀ Values, nM (% NPY-produced displacement) Peptide ¹²⁵I-PYY¹²⁵I-PYY₃₋₃₆ human NPY 0.82 (100%)  1.5 (100%) human NPY₂₋₃₆ 2.3 (100%) 1.2 (100%) human 0.21 (44%) 0.38  (27%) [Leu³¹, Pro³⁴]NPY 340 (56%) 250 (73%) human PYY 1.3 (100%)  0.29 (100%) human PP 11 (20%) untested

Based on the above data, a rat hypothalamic cDNA library of 3×10⁶independent recombinants with a 2.7 kb average insert size wasfractionated into 450 pools of ≈7500 independent clones. All pools weretested in a binding assay with ¹²⁵I-PYY as previously described (U.S.Ser. No. 08/192,288). Seven pools gave rise to positive cells in thescreening assay (#'s 81, 92, 147, 246, 254, 290, 312). Since Y1, Y2, Y4and Y5 receptor subtypes (by PCR or binding analysis) are expressed inrat hypothalamus, the DNA of positive pools were analyzed by PCR withrat Y1, Y2 and Y4 specific primers. Pools # 147, 246, 254 and 312 turnedout to contain cDNAs encoding a Y1 receptor; pool # 290 turned out tocontain cDNA encoding a Y2 receptor subtype; but pools # 81 and 92 werenegative by PCR analysis for Y1, Y2 and Y4 and therefore likelycontained a cDNA encoding a new rat hypothalamic NPY receptor (Y5).Pools # 81 and 92 later turned out to contain an identical NPY receptorcDNA. Pool 92 was subjected to sib selection until a single clone wasisolated (designated CG-18).

The isolated clone carries a 2.8 kb cDNA. This cDNA contains an openreading frame between nucleotides 779 and 2146 that encodes a 456 aminoacid protein. The long 5′ untranslated region could be involved in theregulation of translation efficiency or mRNA stability. The flankingsequence around the putative initiation codon does not conform to theKozak consensus sequence for optimal translation initiation (Kozak,1989, 1991). The hydrophobicity plot displayed seven hydrophobic,putative membrane spanning regions which makes the rat hypothalamic Y5receptor a member of the G-protein coupled superfamily. The nucleotideand deduced amino acid sequences are shown in FIGS. 3 and 4,respectively. Like most G-protein coupled receptors, the Y5 receptorcontains consensus sequences for N-linked glycosylation, in the aminoterminus (position 21 and 28) involved in the proper expression ofmembrane proteins (Kornfeld and Kornfeld, 1985). The Y5 receptor carriestwo highly conserved cysteine residues in the first two extracellularloops that are believed to form a disulfide bond stabilizing thefunctional protein structure (Probst et al, 1992). The Y5 receptor shows9 potential phosphorylation sites for protein kinase C in positions 204,217, 254, 273, 285, 301, 328, 336 and 409 and 2 cAMP- and cGMP-dependentprotein kinase phosphorylation sites in positions 298 and 370. It shouldbe noted that 8 of these 11 potential phosphorylation sites are locatedin the third intra-cellular loop, two in the second intracellular loop,and one in the carboxy terminus of the receptor and could therefore playa role in regulating functional characteristics of the Y5 receptor(Probst et al, 1992). In addition, the rat Y5 receptor carries a leucinezipper motif in its first putative transmembrane domain (Landschulz etal, 1988). A tyrosine kinase phosphorylation site is found in the middleof the leucine zipper.

Localization studies (see below) show that the Y5 mRNA is present inseveral areas of the rat hippocampus. Assuming a comparable localizationin human brain, a human hippocampal cDNA library was screened with ratoligonucleotide primers which were shown to yield a DNA band of theexpected size in a PCR reaction run on human hippocampal cDNA (C.Gerald, unpublished results). Using this PCR screening strategy (Gerald,Adham, Kao, et al., 1995), three positive pools were identified. One ofthese pools was analyzed further, and an isolated clone was purified bysib selection. The isolated clone (CG-19) turned out to contain a fulllength cDNA cloned in the correct orientation for functional expression(see below). The human Y5 nucleotide and deduced amino acid sequencesare shown in FIGS. 5 and 6, respectively. The longest open reading frameencodes a 455 amino acid protein. When compared to the rat Y5 receptorthe human sequence shows 84.1% nucleotide identity (FIGS. 7A to 7E) and87.2% amino acid identity (FIGS. 7F and 7G). The rat protein sequence isone amino acid longer at the very end of both amino and carboxy tails ofthe receptor when compared to the human protein sequence. The human 5-6loop is one amino acid longer than the rat and shows multiple nonconservative substitutions. Even though the 5-6 loops show significantchanges between the rat and human homologs, all of the protein motifsfound in the rat receptor are present in the human homolog. All putativetransmembrane domains and extra cellular loop regions are highlyconserved (FIGS. 7F and 7G). Therefore, both pharmacological profilesand functional characteristics of the rat and human Y5 receptor subtypehomologs may be expected to match closely.

When the human and rat Y5 receptor sequences were compared to other NPYreceptor subtypes or to other human G protein-coupled receptor subtypes,both overall and transmembrane domain identities were very low, showingthat the Y5 receptor genes are not closely related to any otherpreviously characterized cDNAs (Table 4). Even among the human NPYreceptor family, Y1, Y2, Y4 and Y5 members show unusually low levels ofamino acid identity (FIGS. 8A through 8C).

TABLE 4 Human Y5 transmembrane domains identity with other human NPYreceptor subtypes and other human G-protein coupled receptors Receptorsubtype % TM identity Y-4 40 Y-2 42 Y-1 42 MUSGIR 32 DroNPY 31 Beta-1 30Endothelin-1 30 Dopamine D2 29 Adenosine A2b 28 Subst K 28 Alpha-2A 275-HT1Dalpha 26 Alpha-1A 26 IL-8 26 5-HT2 25 Subst P 24

It was also discovered, by PCR using Y5-specific primers, that the humanneuroblastoma cell line SK-N-MC contains Y5 receptor mRNA, butY5-specific binding and functional assays (using agonists) with the cellline were negative. However, a cDNA encoding a functional Y5 receptorwas isolated by PCR from the SK-N-MC cell line.

Northern Blot Analysis

Using the rat Y5 probe, northern hybridizations reveal a strong signalat 2.7 kb and a weak band at 8 kb in rat whole brain. A weak signal isobserved at 2.7 kb in testis. No signal was seen in heart, spleen, lung,liver, skeletal muscle and kidney after a three day exposure (FIG. 16A).This is in agreement with the 2.7 kb cDNA isolated by expression cloningfrom rat hypothalamus and indicates that the disclosed cDNA clone isfull length. The 8 kb band seen in whole brain probably corresponds tounspliced pre-mRNA.

With the human Y5 probe, northern hybridizations (FIGS. 16B and 16C)showed a strong signal at 3.5 kb with a much weaker band at 2.2 and 1.1kb in caudate nucleus, putamen and cerebral cortex, a medium signal infrontal lobe and amygdala and a weak signal in hippocampus, occipitaland temporal lobes, spinal cord, medulla, thalamus, subthalamic nucleus,and substantia nigra. No signal at 3.5 kb was detectable in cerebellumor corpus callosum after a 48 h exposure. It should be noted thatClontech's MTN II and III blots do not carry any mRNA from hypothalamus,periaqueductal gray, superior colliculus and raphe.

Southern blot analysis on human genomic DNA reveals a single bandpattern in 4 of the 5 restriction digests (FIG. 17A). The two bandsobserved in the PstI digest can be explained by the presence of a PstIsite in the coding region of the human Y5 gene. Rat southern blottinganalysis showed a single band pattern in all five restriction digeststested (FIG. 17B). These analyses are consistent with the human and ratgenomes containing a single copy of the Y5 receptor gene.

Canine Y5 Homolog

The longest open reading frame in the canine (beagle) Y5 cDNA (BO11)encodes a 456 amino acid protein with an estimated molecular weight of50 kD. The full-length deduced canine Y5 amino acid sequence is shown inFIG. 24. The canine Y5 receptor is the same length as the rat Y5receptor, and is one amino acid longer than the human Y5 receptor. Thecanine Y5 receptor has 94.3% amino acid identity and 91.7% nucleotideidentity with the human Y5 receptor. The canine Y5 receptor has 91.6%amino acid identity and 82.8% nucleotide identity with the rat Y5receptor. Evidence was found for a potential allelic variation in thebeagle Y5 receptor. In clones BO11 and BO12 there is a T in position477, while in clone BO10 and two partial cDNAs, Bgldog5 and Bgldog6,there is a C in this position. Either nucleotide at this positionresults in an asparagine. Given the high degree of sequence identityamong the three species homologues, the pharmacological profile of thecanine Y5 receptor subtype is expected to closely resemble the human andrat Y5 profiles.

Binding Studies

The cDNA for the rat hypothalamic Y5 receptor was transiently expressedin COS-7 cells for full pharmacological evaluation. ¹²⁵I-PYY boundspecifically to membranes from COS-7 cells transiently transfected withthe rat Y5 receptor construct. The time course of specific binding wasmeasured in the presence of 0.08 nM ¹²⁵I-PYY at 30° C. (FIG. 9). Theassociation curve was monophasic, with an observed association rate(K_(obs)) of 0.06 min⁻¹ and a t_(½) of 11 min; equilibrium binding was99% complete within 71 min and stable for at least 180 min. Allsubsequent binding assays were carried out for 120 min at 30° C. Thebinding of ¹²⁵I-PYY to transiently expressed rat Y5 receptors wassaturable over a radioligand concentration range of 0.4 pM to 2.7 nM.Binding data were fit to a one-site binding model with an apparent K_(d)of 0.29 nM (pK_(d)=9.54±0.13, n=4). A receptor density of between 5 and10 pmol/mg membrane protein was measured on membranes which had beenfrozen and stored in liquid nitrogen (FIG. 10). Membranes frommock-transfected cells, when prepared and analyzed in the same way asthose from CG-18-transfected cells, displayed no specific binding of¹²⁵I-PYY (data not shown). Applicants conclude that the ¹²⁵I-PYY bindingsites observed under the described conditions were derived from the ratY5 receptor construct.

A closely related peptide analog, porcine ¹²⁵I-]Leu³¹, Pro³⁴]PYY, alsobound specifically to membranes from COS-7 cells transiently transfectedwith rat Y5 receptor cDNA. The time course of specific binding wasmeasured at room temperature in both standard binding buffer ([Na⁺]=10mM) and isotonic binding buffer ([Na⁺]=138 mM) using 0.08 nM¹²⁵I-[Leu³¹, Pro³⁴]PYY (FIG. 18). The association curve in 10 mM [Na⁺]was monophasic, with an observed association rate (K_(obs)) of 0.042min⁻¹ and a t_(½) of 17 min; equilibrium binding was 99% complete within110 min and stable for at least 210 min (specific binding was maximal at480 fmol/mg membrane protein). The association curve in 138 mM [Na⁺] wasalso monophasic with a slightly slower time course: (K_(obs)) of 0.029min⁻¹ and a t_(½) of 24 min.; equilibrium binding was 99% completewithin 160 min. and stable for at least 210 min. (specific binding wasmaximal at 330 fmol/mg membrane protein). Note that the specific bindingwas reduced as [Na⁺] was increased; a similar phenomenon has beenobserved for other G protein coupled receptors and may reflect a generalproperty of this receptor family to be modulated by Na⁺ (Horstman et.al., 1990). Saturation binding studies were performed with ¹²⁵I-[Leu³¹,Pro³⁴]PYY in isotonic buffer at room temperature over a 120 minuteperiod. Specific binding to transiently expressed rat Y5 receptors wassaturable over a radioligand concentration range of 0.6 pM to 1.9 nM.Binding data were fit to a one-site binding model with an apparent K_(d)of 0.072 nM (pKd=10.14+0.07, n=2). A receptor density of 560±150 pmol/mgon membranes which had been frozen and stored in liquid nitrogen. That¹²⁵I-[Leu³¹, Pro³⁴]PYY can bind to the rat Y5 receptor with highaffinity at room temperature in isotonic buffer makes it a potentiallyuseful ligand for characterizing the native Y5 receptor in rat tissuesusing autoradiographic techniques. Care must be taken, however, to useappropriate masking agents to block potential radiolabeling of otherreceptors such as Y1 and Y4 receptors (note in Table 6 that rat Y1 andY4 bind the structural homolog [Pro³⁴]PYY). Previously published reportsof ¹²⁵I-[Leu³¹, Pro³⁴]PYY as a Y1-selective radioligand should bere-evaluated in light of new data obtained with the rat Y5 receptor(Dumont et al., 1995).

The pharmacological profile of the rat Y5 receptor was first studied byusing pancreatic polypeptide analogs in membrane binding assays. Therank order of affinity for selected compounds was derived fromcompetitive displacement of ¹²⁵I-PYY (FIG. 11). The rat Y5 receptor wascompared with cloned Y1, Y2, and Y4 receptors from human (Table 5) andrat (Table 6), all expressed transiently in COS-7 cells. One receptorsubtype absent from our panel was the Y3, human or rat, as no modelsuitable for radioligand screening has yet been identified.

TABLE 5 Pharmacological Profile of the Rat Y5 Receptor vs. Y-typeReceptors Cloned from Human

Binding data reflect competitive displacement of ¹²⁵I-PYY from membranesof COS-7 cells transiently expressing rat Y5 and human subtype clones.Peptides were tested at concentrations ranging from 0.001 nM to 1000 nMunless noted. IC₅₀ values corresponding to 50% displacement weredetermined by nonlinear regression analysis and converted to K_(i)values according to the Cheng-Prusoff equation. The data shown arerepresentative of at least two independent experiments.

TABLE 5 K_(i) Values (nM) Peptide Rat Y5 Human Y4 Human Y1 Human Y2rat/human NPY 0.68 2.2 0.07 0.74 porcine NPY 0.66 1.1 0.05 0.81 humanNPY₂₋₃₆ 0.86 16 3.9 2.0 porcine NPY₂₋₃₆ 1.2 5.6 2.4 1.2 porcine NPY₁₃₋₃₆73 38 60 2.5 porcine NPY₂₆₋₃₆ >1000 304 >1000 380 porcine C2-NPY 470 12079 3.5 human [Leu³¹, 1.0 1.1 0.17 >130 Pro³⁴]NPY human [D-Trp³²]-53 >760 >1000 >1000 NPY human NPY free 480 >1000 490 >1000 acidrat/porcine PYY 0.64 0.14 0.35 1.26 human PYY 0.87 0.87 0.18 0.36 humanPYY₃₋₃₆ 8.4 15 41 0.70 human PYY₁₃₋₃₆ 190 46 33 1.5 human [Pro³⁴]- 0.520.12 0.14 >310 PYY human PP 5.0 0.06 77 >1000 human PP₂₋₃₆* not tested0.06 >40 >100 human PP₁₃₋₃₆* not tested 39 >100 >100 rat PP 180 0.16450 >1000 salmon PP 0.31 3.2 0.11 0.17 *Tested only up to 100 nM.

TABLE 6 Pharmacological Profile of the Rat Y5 Receptor vs. Y-typeReceptors Cloned from Rat

Binding data reflect competitive displacement of ¹²⁵I-PYY from membranesof COS-7 cells transiently expressing rat Y5 and rat subtype clones.Peptides were tested at concentrations ranging from 0.001 nM to 1000 nM.IC₅₀ values corresponding to 50% displacement were determined bynonlinear regression analysis and converted to K_(i) values according tothe Cheng-Prusoff equation. The data shown are representative of atleast two independent experiments. Exception: new peptides (marked witha double asterisk) were tested in one or more independent experiments.

TABLE 6 K_(i) Values (nM) Peptide Rat Y5 Rat Y4 Rat Y1 Rat Y2 rat/humanNPY 0.68 1.7 0.12 1.3 porcine NPY ** 0.66 1.78 0.06 1.74 frog NPY **(melano- 0.71 0.09 0.65 statin) human NPY₂₋₃₆ 0.86 5.0 12 2.6 porcineNPY₂₋₃₆ ** 1.1 18 1.6 1.6 porcine NPY₃₋₃₆ ** 7.7 36 91 3.7 porcineNPY₁₃₋₃₆ 73 140 190 31 porcine NPY₁₆₋₃₆ ** 260 200 140 35 porcineNPY₁₈₋₃₆ ** >1000 470 12 porcine NPY₂₀₋₃₆ ** >100 360 93 porcineNPY₂₂₋₃₆ ** >1000 >1000 54 porcine NPY₂₆₋₃₆ ** >1000 >1000 >830 human[Leu³¹, 1.0 0.59 0.10 >1000 Pro³⁴]NPY porcine ** [Leu³¹ , 1.6 0.32 0.25840 Pro³⁴]NPY human (O-Methyl- 1.6 2.3 Tyr²¹)NPY ** human NPY free acid** >610 >1000 720 >980 porcine C2-NPY ** >260 22 140 2.6 humanNPY₁₋₂₄ >1000 >320 >1000 amide ** human [D-Trp³²]NPY 35 >630 >1000 760rat/porcine PYY 0.64 0.58 0.21 0.28 human PYY ** 0.87 0.12 0.30 humanPYY₃₋₃₆ ** 8.4 15 0.48 human PYY₁₃₋₃₆ ** 290 130 14 human [Pro³⁴]PYY0.52 0.19 0.25 >1000 porcine [Pro³⁴]PYY ** 0.64 0.24 0.07 >980 avian PP** >930 >81 >320 >1000 human PP 5.0 0.04 43 >1000 human PP₁₃₋₃₆ **84 >1000 >650 human PP₃₁₋₃₆ ** >1000 26 >10000 >10000 human PP₃₁₋₃₆ freeacid >10,000 >100 ** bovine PP ** 8.4 0.19 120 >1000 frog PP (ranatemporaria) >550 >1000 720 >980 ** rat PP 230 0.19 350 >1000 salmon PP0.33 3.0 0.30 0.16 PYX-1 ** 920 PYX-2 ** >1000 FLRF-amide ** 5500 45000FMRF-amide ** 18000 W(nor-L)RF-amide ** 8700

The rat Y5 receptor possessed a unique pharmacological profile whencompared with human and rat Y-type receptors. It displayed a preferencefor structural analogs of rat/human NPY (K_(i)=0.68 nM) and rat/porcinePYY (K_(i)=0.64 nM) over most PP derivatives. The high affinity forsalmon PP (K_(i)=0.31 nM) reflects the close similarity between salmonPP and rat NPY, sharing 81% of their amino acid sequence and maintainingidentity at key positions: Tyr¹, Gln³⁴, and Tyr⁶. Both N- and C-terminalpeptide domains are apparently important for receptor recognition. TheN-terminal tyrosine of NPY or PYY could be deleted without anappreciable loss in binding affinity (K_(i)=0.86 nM for rat/humanNPY₂₋₃₆), but further N-terminal deletion was disruptive (K_(i)=73 nMfor porcine NPY₁₃₋₃₆). A similar structure-activity relationship wasobserved for PYY and N-terminally deleted fragments such as PYY₃₋₃₆ andPYY₁₃₋₃₆ This pattern places the binding profile of the Y5 receptorsomewhere between that of the Y2 receptor (which receptor can withstandextreme N-terminal deletion) and that of the Y1 receptor (which receptoris sensitive to even a single-residue N-terminal deletion). Note thatthe human Y4 receptor can be described similarly (K_(i)=0.06 nM forhuman PP, 0.06 nM for human PP₂₋₃₆, and 39 nM for human PP₁₃₋₃₆) The Y5receptor resembled both Y1 and Y4 receptors in its tolerance for ligandscontaining Pro³⁴ (as in human [Leu³¹, Pro³⁴]NPY, human [Pro³⁴]-PYY, andhuman PP). Interestingly, the rat Y5 receptor displayed a preference forhuman PP (K_(i)=5.0 nM) over rat PP (K_(i)=180 nM). This patterndistinguishes the rat Y5 from the rat Y4 receptor, which binds bothhuman and rat PP with K_(i) values <0.2 nM. Hydrolysis of the carboxyterminal amide to free carboxylic acid, as in NPY free acid, wasdisruptive for binding affinity for the rat Y5 receptor (K_(i)=480 nM).The terminal amide appears to be a common structural requirement forpancreatic polypeptide family/receptor interactions.

Several peptides shown previously to stimulate feeding behavior in ratsbound to the rat Y5 receptor with K_(i)≦5.0 nM. These include rat/humanNPY (K_(i)=0.68 nM), rat/porcine PYY (K_(i)=0.64 nM), rat/human NPY₂₋₃₆(K_(i)=0.86 nM), rat/human [Leu³¹, Pro³⁴]NPY (K_(i)=1.0 nM), and humanPP (K_(i)=5.0 nM). conversely, peptides which were relatively lesseffective as orexigenic agents bound weakly to CG-18. These includeporcine NPY₁₃₋₃₆ (K_(i)=73 nM), porcine C2-NPY (K_(i)=470 nM) and humanNPY free acid (K_(i)=480 nM). The rank order of K_(i) values are inagreement with rank orders of potency and activity for stimulation offeeding behavior when peptides are injected i.c.v. or directly into rathypothalamus (Clark et al., 1984; Stanley et al., 1985; Kalra et al.,1991; Stanley et al., 1992). The rat Y5 receptor also displayed moderatebinding affinity for [D-Trp³²]NPY (K_(i)=53 nM), the modified peptidereported to regulate NPY-induced feeding by Balasubramaniam et al.(1994). It is noteworthy that [D-Trp³²]NPY was ≧10-fold selective forCG-18-over the other cloned receptors studied, whether human or rat.These data clearly and definitively link the cloned Y5 receptor to thefeeding response.

The cDNA corresponding to the human Y5 homolog isolated from humanhippocampus was transiently expressed in COS-7 cells for membranebinding studies. The binding of ¹²⁵I-PYY to the human Y5 receptor(CG-19) was saturable over a radioligand concentration range of 8 pM to1.8 nM. Binding data were fit to a one-site binding model with anapparent K_(d) of 0.10 nM in the first experiment. Repeated testingyielded an apparent K_(d) of 0.18 nM (PK_(d)=9.76±0.11, n=4). A maximumreceptor density of 500 fmol/mg membrane protein was measured on freshmembranes. As determined by using peptide analogs within the pancreaticpolypeptide family, the human Y5 pharmacological profile bears astriking resemblance to the rat Y5 receptor (Tables 7 and 8).

TABLE 7 Pharmacological Profile of the Rat Y5 Receptor vs. the Human YSReceptor, as Expressed both Transiently in COS-7 and Stably in LM(tk-)cells

Binding data reflect competitive displacement of radioligand (either¹²⁵I-PYY or ¹²⁵I-PYY₃₋₃₆ as indicated) from membranes of COS-7 cellstransiently expressing the rat Y5 receptor and its human homolog or fromLM(tk-) cells stably expressing the human Y5 receptor. Peptides weretested at concentrations ranging from 0.001 nM to 1000 nM. IC₅₀ valuescorresponding to 50% displacement were determined by nonlinearregression analysis and converted to K_(i) values according to theCheng-Prusoff equation. New peptides are marked with a double asterisk.

TABLE 7 K_(i) Values (nM) Rat Y5 Human Y5 Human Y5 Human Y5 (COS-7,(COS-7, (LM(tk-), (LM(tk-), Peptide ¹²⁵I-PYY) ¹²⁵I-PYY) ¹²⁵I-PYY)¹²⁵I-PYY₃₋₃₆) rat/human NPY 0.68 0.15 0.89 0.65 porcine NPY 0.68 1.4 **human 0.86 0.33 1.6 0.51 NPY₂₋₃₆ porcine 0.66 0.58 1.2 NPY₂₋₃₆ **porcine 73 110 39 NPY₁₃₋₃₆ porcine 260 300 180 NPY₁₆₋₃₆ **porcine >1000 >470 310 NPY₁₈₋₃₆ ** porcine >1000 >1000 NPY₂₂₋₃₆ **porcine >1000 >1000 NPY₂₆₋₃₆ ** human [Leu³¹, 1.0 0.72 3.0 Pro³⁴]NPYhuman [Leu³¹, 2.4 1.4 Pro³⁴]NPY ** human NPY >610 >840 free acid **porcine 260 370 260 220 C2-NPY ** human 35 35 16 10 [D-Trp³²]NPYrat/porcine 0.64 0.75 PYY human PYY ** 0.87 0.44 1.3 0.43 human 8.4 178.1 1.6 PYY₃₋₃₆ ** human 0.52 0.34 1.7 1.7 [Pro³⁴]PYY human PP 5.0 1.73.0 1.2 human 2.1 PP₂₋₃₆ ** human 290 720 PP₁₃₋₃₆ ** human >10000 >1000041000 PP₃₁₋₃₆ ** human [Ile³¹, 2.0 Gln³⁴]PP ** bovine PP ** 8.4 1.6 7.95.0 rat PP 230 630 130 salmon PP 0.33 0.27 0.63

TABLE 8 Pharmacological Profile of the Human Y5 Receptor vs. Y-typeReceptors Cloned from Human

Binding data reflect competitive displacement of ¹²⁵I-PYY from membranesof COS-7 cells transiently expressing human Y5 other sub-type clones.Peptides were tested at concentrations ranging from 0.001 nM to 1000 nMunless noted. IC₅₀ values corresponding to 50% displacement weredetermined by nonlinear regression analysis and converted to K_(i)values according to the Cheng-Prusoff equation. The data shown arerepresentative of at least two independent experiments.

TABLE 8 K_(i) Values (nM) Peptide Human Y5 Human Y4 Human Y1 Human Y2rat/human NPY 0.46 2.2 0.07 0.74 porcine NPY 0.68 1.1 0.05 0.81 human0.75 16 3.9 2.0 NPY₂₋₃₆ porcine 0.58 5.6 2.4 1.2 NPY₂₋₃₆ porcine 110 3860 2.5 NPY₁₃₋₃₆ porcine >1000 304 >1000 380 NPY₂₆₋₃₆ porcine C2- 370 12079 3.5 NPY human [Leu³¹, 1.6 1.1 0.17 >130 Pro³⁴]NPY human35 >760 >1000 >1000 [D-Trp³²]NPY human NPY >840 >1000 490 >1000 freeacid rat/porcine 0.58 0.14 0.35 1.26 PPY human PYY 0.44 0.87 0.18 0.36human 17 15 41 0.70 PYY₃₋₃₆ human not tested 46 33 1.5 PPY₁₃₋₃₆ human0.77 0.12 0.14 >310 [Pro³⁴]PYY human PP 1.4 0.06 77 >1000 human PP₂₋₃₆*2.1 0.06 >40 >100 human 720 39 >100 >100 PP₁₃₋₃₆* rat PP 630 0.16450 >1000 salmon PP 0.46 3.2 0.11 0.17 *Tested only up to 100 nM.

Binding Studies of hY5 Expressed in Insect Cells

Tests were initially performed to optimize expression of hY5 receptor.Infecting Sf9, Sf21, and High Five cells with hY5BB3 virus at amultiplicity of infection (MOI) of 5 and preparing membranes for bindinganalyses at 45 hrs. postinfection, B_(max) ranges from 417 to 820fmoles/mg protein, with the highest expression being hY5BB3 in Sf21cells were observed. Therefore, the next series of experiments used Sf21cells. Optimal multiplicity of infection (the ratio of viral particlesto cells) was next examined by testing MOI of 1, 2, 5 and 10. TheB_(max) values were ≈1.1-1.2 pmoles/mg protein for any of the MOIs,suggesting that increasing the number of viral particles per cell isneither deleterious nor advantageous. Since viral titer calculations areapproximate, MOI=5 was used for future experiments. The last parametertested was hours postinfection for protein expression, ranging from45-96 hours postinfection. It was found that optimal expression occurred45-73 hrs. postinfection. In summary, a hY5 recombinant baculovirus hasbeen created which binds ¹²⁵I-PYY with a B_(max) of ≈1.2 pmoles/mgprotein.

Human Y5 Homolog: Transient Expression in Baculovirus-Infected Sf21Insect Ovary Cells

Sf21 cells infected with a human Y5 baculovirus construct were harvestedas membrane homogenates and screened for specific binding of ¹²⁵I-PYYusing 0.08 nM radioligand. Specific binding was greatest (500 fmol/mgmembrane protein) for sample D-2/[4], derived from Sf-21 cells. Nospecific binding was observed after infection with the baculovirusplasmid alone (data not shown). If the assumption is made that thebinding affinity of porcine ¹²⁵I-PYY for the human Y5 receptor is thesame whether the expression system is COS-7 or baculovirus/Sf-21 (0.18nM), the specific binding in sample D-2/[4] predicts an apparent B_(max)of 1600 fmol/mg membrane protein. The Y5 receptor yield in thebaculovirus/Sf21 expression system is therefore as good or better thanthat in COS-7. We conclude that the baculovirus offers an alternativetransfection technique amenable to large batch production of the humanY5 receptor.

Binding Studies Using the Canine Y5 Receptor

Membranes from COS-7 cells transiently transfected with canine Y5receptor (using the plasmid designated cY5-BO11, ATCC Accession No.97587) displayed specific binding of porcine ¹²⁵I-PYY. The binding wassaturable over a concentration range of 0.6 pM to 2.7 nM, with anobserved K_(d) of 1.1 nM and a B_(max) of 5700 fmol/mg membrane protein.Compounds selected for the ability to bind or activate the human and ratY5 receptor homologs were subsequently tested for binding to the canineY5 receptor (Table 20). The pharmacological profile for the canine Y5receptor was in general agreement with those derived for the otherspecies homologs. For example, the canine Y5 receptor bound human NPY,PYY and PP with K_(i) values <10 nM. The canine Y5 receptor bound bovinePP with higher affinity (10 nM) than rat PP (160 nM), as is also thecase for the rat and human Y5 receptor homologs. Binding affinity wasnot disturbed by substitution of Gln³⁴ in NPY or PYY with Pro³⁴ (as in[Leu³¹, Pro³⁴]NPY, K_(i)=4.1 or [Pro³⁴]PYY, K_(i)=1.4 nM). In thisregard, the canine Y5 receptor exhibits what has been historicallyperceived as a Y1-like property. It was also observed that deletion ofTyr¹ from NPY (as in NPY₂₋₃₆) was not disruptive (K_(i)=2.1 nM). Furtherdeletion of NPY and PYY to fragments such as NPY₃₋₃₆, PYY₃₋₃₆ andNPY₁₃₋₃₆, however, was increasingly disruptive. The canine Y5 receptorbound the Y2-selective and centrally modified analog C2-NPY withrelatively weak affinity (K_(i)=300 nM). It is concluded that the canineY5 receptor, like the rat and human Y5 counterparts, depends on selectedresidues in the N-terminal, central and C-terminal regions of the parentpeptide for optimal binding affinity. Particularly diagnostic tools suchas the Y5-selective peptide D-[Trp³²]NPY and the Y1-selective antagonistBIBP 3226 (Rudolf, et al., 1994) were bound by the canine Y5 receptorwith K_(i) values of 35 and 17000 nM, respectively. These values are inthe range of those reported for the rat and human Y5 homologs.

BIBP 3226 was also tested for binding affinity at the cloned humanY-type receptors, and was observed to bind with K_(i) values of 14 nMfor the Y1 receptor, 6900 nM for the Y2 receptor, 8000 nM for the Y4receptor and 49000 nM for the Y5 receptor. Similar experiments withcloned rat Y-type receptors generated K_(i) values of 20 nM for the Y1receptor, 66000 nM for the Y2 receptor, 420 nM for the Y4 receptor and25000 nM for the Y5 receptor. BIBP 3226 blocked NPY-induced activationof rat Y1 receptors with a K_(b) of 9.4 nM and also blocked PP-inducedactivation of rat Y4 receptors with a Kb of 4800 uM; there was noevidence for antagonism of NPY- or PP-induced activation of rat Y2 or Y5receptors at concentrations up to 1 μM. These data further confirm theclassification of BIBP 3226 as a Y1-selective receptor antagonist.

Stable Expression Systems for Y5 Receptors: Characterization in BindingAssays

The cDNA for the rat Y5 receptor was stably transfected into 293 cellswhich were pre-screened for the absence of specific ¹²⁵I-PYY binding(data not shown). After co-transfection with the rat Y5 cDNA plus aG-418-resistance gene and selection with G-418, surviving colonies werescreened as membrane homogenates for specific binding of ¹²⁵I-PYY using0.08 nM radioligand. A selected clone (293 clone # 12) bound 65 fmol¹²⁵I-PYY/mg membrane protein and was isolated for further study infunctional assays.

The cDNA for the human Y5 receptor was stably transfected into bothNIH-3T3 and LM(tk-) cells, each of which were pre-screened for theabsence of specific ¹²⁵I-PyY binding (data not shown). Afterco-transfection with the human Y5 cDNA plus a G-418-resistance gene andselection with G-418, surviving colonies were screened as membranehomogenates for specific binding of ¹²⁵I-PYY using 0.08 nM radioligand.NIH-3T3 clone #8 bound 46 fmol ¹²⁵I-PYY/mg membrane protein and LM(tk-)clone #7 bound 32 fmol ¹²⁵I-PYY/mg membrane protein. These two cloneswere isolated for further characterization in binding and cAMPfunctional assays. A third clone which bound 25 fmol/mg membraneprotein, LM(tk-) #3, was evaluated in calcium mobilization assays.

The human Y5 stably expressed in NIH-3T3 cells (clone #8) was furthercharacterized in saturation binding assays using ¹²⁵I-PYY. The bindingwas saturable over a concentration range of 0.4 pM to 1.9 nM. Bindingdata were fit to a one-site binding model with an apparent K_(d) of 0.30nM (PK_(d)=9.53, n=1) and an apparent B_(max) of 2100 fmol/mg membraneprotein using fresh membranes.

The human Y5 stably expressed in LM(tk-) cells (clone #7) was furthercharacterized in saturation binding assays using ¹²⁵I-PYY, ¹²⁵I-PYY₃₋₃₆,and ¹²⁵I-NPY. ¹²⁵I-PYY binding was saturable according to a 1-site modelover a concentration range of 0.4 pM to 1.9 nM, with an apparent K_(d)of 0.47 nM (pK_(d)=9.32±0.07, n=5) and an apparent B_(max) of up to 8pmol/mg membrane protein when membranes had been frozen and stored inliquid nitrogen. Peptide K_(i) values derived from ¹²⁵I-PYY binding tohuman Y5 receptors from LM(tk-) were comparable to those derived fromthe previously described human and rat Y5 expression systems (Table 7).¹²⁵I-PYY₃₋₃₆ binding to the human Y5 in LM(tk-) cells was also saturableaccording to a 1-site model over a concentration range of 0.5 pM to 2.09nM, with an apparent K_(d) of 0.40 nM (PK_(d)=9.40, n=1) and an apparentB_(max) of 490 fmol/mg membrane protein when membranes had been frozenand stored in liquid nitrogen. Peptide ligands appeared to bind withcomparable affinity to human Y5 receptors in LM(tk-) cells whether theradioligand used was ¹²⁵I-PYY or ¹²⁵I-PYY₃₋₃₆ (Table 7). Finally,¹²⁵I-NPY binding to the human Y5 in LM(tk-) cells was saturableaccording to a 1-site model over a concentration range of 0.4 pM to 1.19nM, with an apparent K_(d) of 0.28 and an apparent B_(max) of 360fmol/mg membrane protein when membranes had been frozen and stored inliquid nitrogen.

Considering the saturation binding studies for the human and rat Y5receptor homologs as a whole, the data provide evidence that the Y5receptor is a target for multiple radioiodinated peptide analogs in thepancreatic polypeptide family, including ¹²⁵I-PYY, ¹²⁵I-NPY,¹²⁵I-PYY₃₋₃₆ and ¹²⁵I-[Leu³¹, Pro³⁴]PYY. The so-called Y1 andY2-selective radioligands (such as ¹²⁵I-[Leu³¹, Pro³⁴]PYY and¹²⁵I-PYY₃₋₃₆, respectively (Dumont et al., 1995)) should be used withcaution when probing native tissues for Y-type receptor expression.

Receptor/G protein Interactions: Effects of Guanine Nucleotides

For a given G protein-coupled receptor, a portion of the receptorpopulation can typically be characterized in the high affinity ligandbinding site using discriminating agonists. The binding of GTP or anon-hydrolyzable analog to the G protein causes a conformational changein the receptor which favors a low affinity ligand binding state.Whether the non-hydrolyzable GTP analog, Gpp(NH)p, would alter thebinding of ¹²⁵I-PYY to Y5 in COS-7 and LM(tk-) cells (FIG. 19) wasinvestigated. ¹²⁵I-PYY binding to both human and rat Y5 receptors inCOS-7 cells was relatively insensitive to increasing concentrations ofGpp(NH)p ranging from 1 nM to 100 μM (FIG. 19), as was also the case fordog Y5 receptors in COS-7 cells (data not shown). The human Y5 receptorin LM(tk-) cells, however, displayed a concentration dependent decreasein radioligand binding (−85 fmol/mg membrane protein over the entireconcentration range). The difference between the receptor preparationscould be explained by several factors, including 1) the types of Gproteins available in the host cell for supporting a high affinityreceptor-agonist complex, 2) the level of receptor reserve in the hostcell, 3) the efficiency of receptor/G protein coupling, and 4) theintrinsic ability of the agonist (in this case, ¹²⁵I-PYY) to distinguishbetween multiple conformations of the receptor.

Functional Assay

Activation of all Y-type receptors described thus far is thought toinvolve coupling to pertussis toxin-sensitive G-proteins which areinhibitory for adenylate cyclase activity (G_(i) or G_(o)) (Wahlestedtand Reis, 1993). That the atypical Y1 receptor is linked to cyclaseinhibition was prompted by the observation that pertussis toxininhibited NPY-induced feeding in vivo (Chance et al., 1989); a moredefinitive analysis was impossible in the absence of the isolatedreceptor. Based on these prior observations, the ability of NPY toinhibit forskolin-stimulated cAMP accumulation in human embryonic kidney293 cells stably transfected with rat Y5 receptors was investigated.Incubation of intact cells with 10 μM forskolin produced a 10-foldincrease in cAMP accumulation over a 5 minute period, as determined byradioimmunoassay. Simultaneous incubation with rat/human NPY decreasedthe forskolin-stimulated cAMP accumulation by 67% in stably transfectedcells (FIG. 12), but not in untransfected cells (data not shown). It isconcluded that the rat Y5 receptor activation results in decreased cAMPaccumulation, very likely through inhibition of adenylate cyclaseactivity. This result is consistent with the proposed signalling pathwayfor all Y-type receptors and for the atypical Y1 receptor in particular.

Peptides selected for their ability to stimulate feeding behavior inrats were able to activate the rat Y5 receptor with EC₅₀<10 nM (Kalra etal., 1991; Stanley et al., 1992; Balasubramaniam et al., 1994). Theseinclude rat/human NPY (EC₅₀=1.8 nM), rat/human NPY₂₋₃₆ (EC₅₀=2.0 nM),rat/human [Leu³¹, Pro³⁴]NPY (EC₅₀=0.6 nM), rat/porcine PYY (EC₅₀=4.0nM), and rat/human [D-Trp³²]NPY (EC₅₀=7.5 nM) (Table 9). K_(i) valuesderived from rat Y5-dependent binding of ¹²⁵I-PYY and peptide ligands.(Table 5) were in close range of EC₅₀ values derived from ratY5-dependent regulation of cAMP accumulation (Table 9). The maximalsuppression of cAMP produced by all peptides in Table 9 was between 84%and 120% of that produced by human NPY, except in the case of FLRFamide(42%). Of particular interest is the Y5-selective peptide [D-Trp³²]NPY.This is a peptide which was shown to stimulate food intake when injectedinto rat hypothalamus, and which also attenuated NPY-induced feeding inthe same paradigm (Balasubramaniam, 1994). It was observed that[D-Trp³²]NPY bound weakly to other Y-type clones with K_(i)>500 nM(Tables 5 and 6) and displayed no activity in functional assays (Table11). In striking contrast, [D-Trp³²]NPY bound to the rat Y5 receptorwith a K_(i)=53 nM and was fully able to mimic the inhibitory effect ofNPY on forskolin-stimulated cAMP accumulation with an EC₅₀ of 25 nm andan E_(max)=72%. That [D-Trp³²]NPY was able to selectively activate theY5 receptor while having no detectable activity at the other subtypeclones strongly suggests that Y5 receptor activation is responsible forthe stimulatory effect of [D-Trp³²]NPY on feeding behavior in vivo.

TABLE 9 Functional Activation of the Rat Y5 Receptor

Functional data were derived from radioimmunoassay of cAMP accumulationin stably transfected 293 cells stimulated with 10 μM forskolin.Peptides were tested for agonist activity at concentrations ranging from0.03 pM to 0.3 μM. The maximum inhibition of cAMP accumulation (E_(max))and the concentration producing a half-maximal effect (EC₅₀) weredetermined by nonlinear regression analysis according to a 4 parameterlogistic equation. New peptides are marked with a double asterisk.

TABLE 9 Peptide E_(max) EC₅₀ (nM) rat/human 67% 1.8 NPY porcine NPY **0.79 rat/human 84% 2.0 NPY₂₋₃₆ porcine NPY₂₋₃₆ ** 1.2 porcine 21NPY₁₃₋₃₆ ** rat/human 70% 0.6 [Leu³¹, Pro³⁴]NPY porcine 1.1 [Leu³¹,Pro³⁴]NPY ** porcine C2-NPY ** 240 rat/human 72% 9.5 [D-Trp³²]NPYrat/porcine 86% 4.0 PYY human PYY ** 1.5 human PYY₃₋₃₆ ** 4.9 human 1.8[Pro³⁴]PYY ** human PP ** 1.4 bovine PP ** 5.7 salmon PP ** 0.92 rat PP** 130 PYX-1 ** >300 PYX-2 ** >300 FLRFamide ** 13 000

The ability of the human Y5 receptor to inhibit CAMP accumulation wasevaluated in NIH-3T3 and IM(tk-) cells, neither of which display anNPY-dependent regulation of [cAMP] without the Y5 constrict. Intactcells stably transfected with the human Y5 receptor were analyzed asdescribed above for the rat Y5 CAMP assay. Incubation of stablytransfected NIH-3T3 cells with 10 uM forskolin generated an average21-fold increase in [cAMP] (n=2). Simultaneous incubation with human NPYdecreased the forskolin-stimulated [cAMP] with an E_(max) of 42% and anEC₅₀ of 8.5 nM (FIG. 20). The technique of suspending and then replatingthe Y5-transfected LM(tk-) cells was correlated with a robust andreliable cellular response to NPY-like peptides and was thereforeincorporated into the standard methodology for the functional evaluationof the human Y5 in LM(tk-). Incubation of stably transfected LM(tk-)cells prepared in this manner produced an average 7.4-fold increase in[cAMP] (n=87). Simultaneous incubation with human NPY decreased theforskolin-stimulated [cAMP] with an E_(max) of 72% and with an EC₅₀ of2.4 nM (FIG. 20). The human Y5 receptor supported a cellular response toNPY-like peptides in a rank order similar to that described for the ratY5 receptor (Table 6, 10). As the rat Y5 receptor is clearly linked by[D-Trp³²]NPY and other pharmacological tools to the NPY-dependentregulation of feeding behavior, the human Y5 receptor is predicted tofunction in a similar fashion. Both the human and receptor homologsrepresent useful models for the screening of compounds intended tomodulate feeding behavior by interfering with NPY-dependent pathways.

TABLE 10 Functional Activation of the Human Y5 Receptor in a cAMPRadioimmunoassay

Functional data were derived from radioimmunoassay of cAMP accumulationin stably transfected LM(tk-) cells stimulated with 10 μM forskolin.Peptides were tested for agonist activity at concentrations ranging from0.03 pM to 0.3 μM. The maximum inhibition of cAMP accumulation (E_(max))and the concentration producing a half-maximal effect (EC₅₀) weredetermined by nonlinear regression analysis according to a 4 parameterlogistic equation.

TABLE 10 % inhibition relative to Peptide human NPY EC₅₀ (nM) rat/humanNPY 100% 2.7 porcine NPY 107% 0.99 rat/human NPY₂₋₃₆ 116% 2.6 porcineNPY₂₋₃₆  85% 0.71 porcine NPY₁₃₋₃₆ 49 rat/human 3.0 [Leu³¹, Pro³⁴]NPYporcine 1.3 [Leu³¹, Pro³⁴]NPY rat/human [D- 108% 26 Trp³²]NPYrat/porcine PYY 109% 3.6 human PYY 111% 4.9 human PYY₃₋₃₆ 18 human[Pro³⁴]PYY 108% 2.5 human PP 96% 14 human PP₂₋₃₆ 2.0 human 5.6 [Ile³¹,Gln³⁴]PP bovine PP 4.0 salmon PP 96% 4.5

TABLE 11 Binding and Functional Characterization of [D-Trp³²]NPY

Binding data were generated as described in Tables 5 and 6. Functionaldata were derived from radioimmunoassay of cAMP accumulation in stablytransfected cells stimulated with 10 μM forskolin. [D-Trp³²]NPY wastested for agonist activity at concentrations ranging from 0.03 pM to0.3 μM. Alternatively, [D-Trp³²]NPY was included as a single spike (0.3μM) in the human PYY concentration curve for human Y1 and human Y2receptors, or in the human PP concentration curve for human Y4receptors, and antagonist activity was detected by the presence of arightward shift (from EC₅₀ to EC₅₀′). K_(b) values were calculatedaccording to the equation: K_(b)=[[D-Trp³²]NPY/((EC50/EC₅₀′)−1). Thedata shown are representative of at least two independent experiments.

TABLE 11 Binding Function Receptor K_(i) EC₅₀ K_(b) Subtype Species (nM)(nM) (nM) Activity Y1 Human >1000 None detected Y2 Human >1000 Nonedetected Y4 Human >1000 None detected Y5 Human   18 26 Not Determined Y1Rat >1000 Not Determined Y2 Rat >1000 Not Determined Y4 Rat >1000 NotDetermined Y5 Rat   53 9.50 Agonist

Functional Assay: Intracellular Calcium Mobilization

The intracellular free calcium concentration was increased in LM(tk-)cells stably transfected with the human Y5 receptor within 30 seconds ofincubation with 100 nM human NPY (ΔCa²⁺=34 nM, FIG. 21D). UntransfectedLM(tk-) cells did not respond to human NPY (data not shown). The calciummobilization provides a second pathway through which Y5 receptoractivation can be measured. These data also serve to link with the Y5receptor with other cloned human Y-type receptors, all of which havebeen demonstrated to mobilize intracellular calcium in variousexpression systems (FIG. 21).

Localization Studies

The mRNA for the NPY Y5 receptor was widely distributed in rat brain,and appeared to be moderately abundant (Table 12 and FIG. 13). Themidline thalamus contained many neurons with silver grains over them,particularly the paraventricular thalamic nucleus, the rhomboid nucleus,and the nucleus reunions. In addition, moderately intense hybridizationsignals were observed over neurons in both the centromedial andanterodorsal thalamic nuclei. In the hypothalamus, a moderate level ofhybridization signal was seen over scattered neurons in the lateralhypothalamus, paraventricular, supraoptic, arcuate, and dorsomedialnuclei. In both the medial preoptic nucleus and suprachiasmatic nucleus,weak or moderate accumulations of silver grains were present. In thesuprachiasmatic nucleus, hybridization signal was restricted mainly tothe ventrolateral subdivision. In the paraventricular hypothalamus,positive neurons were observed primarily in the medial parvicellularsubdivision.

TABLE 12 Distribution of NPY Y5 mRNA in the Rat CNS REGION Y5 mRNACerebral cortex +1 Thalamus paraventricular n. +3 rhomboid n. +3reunions n. +3 anterodorsal n. +2 Hypothalamus paraventricular n. +2lateral hypoth. area +2/+3 supraoptic n. +1 medial preoptic n. +2suprachiasmatic n. +1/+2 arcuate n. +2 Hippocampus dentate gyrus +1polymorph dentate gyrus +2 CA1  0 CA3 +1 Amygdala central amygd. n.,medial +2 anterior cortical amygd. n. +2 Olivary pretectal n. +3Anterior pretectal n. +3 Substantia nigra, pars compacta +2 Superiorcolliculus +2 Central gray +2 Rostral linear raphe +3 Dorsal raphe +1Inferior colliculus +1 Medial vestibular n. +2/+3 Parvicellular ret. n.,alpha +2 Gigantocellular reticular n., +2 alpha Pontine nuclei +1/+2

Moderate hybridization signals were found over most of the neurons inthe polymorphic region of the dentate gyrus in the hippocampus, whilelower levels were seen over scattered neurons in the CA3 region. In theamygdala, the central nucleus and the anterior cortical nucleuscontained neurons with moderate levels of hybridization signal. In themesencephalon, hybridization signals were observed over a number ofareas. The most intense signals were found over neurons in the anteriorand olivary pretectal nuclei, periaquaductal gray, and over the rostrallinear raphe. Moderate hybridization signals were observed over neuronsin the internal gray layer of the superior colliculus, the substantianigra, pars compacta, the dorsal raphe, and the pontine nuclei. Most ofthe neurons in the inferior colliculus exhibited a low level of signal.In the medulla and pons, few areas exhibited substantial hybridizationsignals. The medial vestibular nucleus was moderately labeled, as wasthe parvicellular reticular nucleus, pars alpha, and the gigantocellularreticular nucleus.

Little or no hybridization signal was observed on sections hybridizedwith the radiolabeled sense oligonucleotide probe. More importantly, inthe transfected COS-7 cells, the antisense probe hybridized only to thecells transfected with the rat Y5 cDNA (Table 13). These resultsindicate that the probe used to characterize the distribution of Y5 mRNAin rat brain is specific for this mRNA, and does not cross-hybridize toany of the other known NPY receptor mRNAs.

TABLE 13 Hybridization of antisense oligonucleotide probes totransfected COS-7 cells. Hybridization was performed as described inMethods. The NPY Y5 probe hybridizes only to the cells transfected withthe Y5 CDNA. ND = not done. Cells Mock rY1 rY2 rY4 rY5 Oligo rY1 − + −ND ND rY2 − − + − − rY4 − − − + − rY5 − − − − +

In vivo Studies with Y5-selective Compounds

The results reported above strongly support a role for the Y5 receptorin regulating feeding behavior. Accordingly, the binding and functionalproperties of several newly synthesized compounds at the cloned humanY1, human Y2, human Y4, and human Y5 receptors was evaluated.

Table 14 discloses several compounds which bind selectively to the humanY5 receptor and act as Y5 receptor antagonists, as measured by theirability to block NPY-induced inhibition of CAMP accumulation inforskolin-stimulated LM(tk-) cells stably transfected with the clonedhuman Y5 receptor. The structures of the compounds described in Table 13are shown in FIG. 22. Preliminary experiments indicate that compound 28is a Y5 receptor antagonist.

TABLE 14 Evaluation of Human Y5 Receptor Antagonists

The ability of the compounds to antagonize the Y-type receptors isreported as the K_(b). The K_(b) is derived from the EC₅₀, orconcentration of half-maximal effect, in the presence (EC₅₀) or absence(EC₅₀′) of compound, according to the equation:K_(b)=[NPY]/((EC₅₀/EC₅₀′)−1). The results shown are representative of atleast three independent experiments. N.D.=Not determined.

TABLE 14 Binding Affinity (K_(i) (nM) vs. ¹²⁵I-PYY) Human ReceptorCompound Y1 Y2 Y4 Y5 K_(b) (nM)  1 1660 1920 4540 38.9 183  2 1806 3861280 17.8 9.6  5 3860 249 2290 1.27 2.1  6 4360 4610 32,900 47.5 93  72170 2870 7050 42.0 105  9 3240 >100,000 3720 108 479 10 1070 >100,0005830 40.7 2.8 11 1180 >100,000 7130 9.66 1.5 17 5550 1000 8020 14 6.0 193550 955 11700 11 23 20 16000 7760 20400 8.3 26 21 13000 1610 18500 9.816 22 17200 7570 27500 11 3.0 23 14500 617 21500 26 38 25 3240 851 1310017 311 26 23700 58200 19300 14 50 27 48700 5280 63100 28 4928 >100,000 >75,000 >100,000 19,000 N.D.

Several of these compounds were further tested using in vivo animalmodels of feeding behavior.

Since NPY is the strongest known stimulant of feeding behavior,experiments were performed with several compounds to evaluate the effectof the compounds described above on NPY-induced feeding behavior insatiated rats.

First, 300 pmole of porcine NPY in vehicle (ACSF) was administered byintracerebroventricular (i.c.v.) injection, along with i.p.administration of compound vehicle (10% DMSO/water), and the food intakeof NPY-stimulated animals was compared to food intake in animals treatedwith the vehicles. The 300 pmole injection of NPY was found tosignificantly induce food intake (p<0.05; Student-Newman-Keuls).

Using the 300 pmole dose of NPY found to be effective to stimulatefeeding, other animals were treated with the compounds byintraperitoneal (i.p.) administration, followed 30-60 minutes later byi.c.v. NPY administration, and measurement of subsequent food intake. Asshown in Table 15, NPY-induced food intake was significantly reduced inanimals first treated with the compounds (p<0.05, Student-Newman-Keuls).These experiments demonstrate that NPY-induced food intake issignificantly reduced by administration to animals of a compound whichis a. Y5-selective antagonist.

TABLE 15

NPY-induced cumulative food intake in rats treated with either thei.c.v. and i.p. vehicles (control), 300 pmole NPY alone (NPY), or inrats treated first with compound and then NPY (NPY+compound). Foodintake was measured 4 hours after stimulation with NPY. Food intake isreported as the mean±S.E.M. intake for a group of animals.

TABLE 15 Food intake (g) mean ± S.E.M. Compound  1  5 17 19 Compound 1010 10 30 Dose (mg/kg i.p.) control 3.7 ± 0.6 2.4 ± 0.5 2.4 ± 0.7 2.9 ±0.8 (vehicles only) NPY 7.4 ± 0.5 6.8 ± 1.0 5.8 ± 0.5 4.9 ± 0.4 NPY +4.6 ± 0.6 4.1 ± 0.4 3.8 ± 0.4 1.5 ± 0.6 compound

Since food deprivation induces an increase in the hypothalamic NPYlevels, it has been postulated that food intake following a period offood deprivation is NPY-mediated. Therefore, the Y5 antagonists of Table14 were administered by intraperitoneal injection at a dose of 30 mg/kgto conscious rats following a 24h food deprivation. The human Y5receptor antagonists shown in Table 14 reduced food intake in thefood-deprived animals, as shown below in Table 16. The food intake ofanimals treated with test compound is reported as the percentage of thefood intake measured for control animals (treated with vehicle), i.e.,25% means the animals treated with the compound consumed only 25% asmuch food as the control animals. Measurements were performed two hoursafter administration of the test compound.

TABLE 16 Two-hour food intake of food-deprived rats. Food intake isexpressed as the percentage of intake compared to control rats. N. D. =Not done. Mean Compound (%)  1 34  2 42  5 87  6 38  7 47  9 40 10 74 1115 17 27 19 36 20 35 21 80 22 55 23 58 25 32 26 73 27 84 28 ND

These experiments indicate that the compounds of the present inventioninhibit food intake in rats, especially when administered in a range ofabout 0.01 to about 100 mg/kg rat, by either oral, intraperitoneal orintravenous administration. The animals appeared normal during theseexperiments, and no ill effects on the animals were observed after thetermination of the feeding experiments.

The binding properties of the compounds were also evaluated with respectto other cloned human G-protein coupled receptors. As shown in Table 17,below, the Y5-selective compounds described hereinabove exhibited loweraffinity for receptors other than the Y-type receptors.

TABLE 17 Cross-reactivity of compounds at other cloned human receptorsReceptor (pKi) Compound α_(1d) α_(1b) α_(1a) α_(2a) α_(2b) α_(2c) H1 H2D3 5HT_(1a) 5HT₂ 5HT₇ 5HT_(1F) 5HT_(1E) 5HT_(1Dβ) 5HT_(1Dα)  1 6.25 6.236.15 6.28 6.01 6.34 5.59 6.32 5.69 4.51 6.34 6.20 5.30 5.30 5.30 5.42  2N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.N.D. N.D.  5 7.24 7.36 7.63 7.39 7.29 7.63 6.65 6.68 7.24 6.33 6.41 6.005.30 5.30 5.55 5.37  6 5.68 5.73 6.54 7.14 5.79 6.35 N.D. N.D. N.D. N.D.N.D. 6.00 5.30 5.30 5.30 5.30  7 6.46 6.08 6.06 7.16 6.09 6.85 N.D. N.D.N.D. N.D. N.D. 6.64 5.30 5.30 5.30 5.85  9 6.45 6.26 6.57 7.04 5.00 6.81N.D. N.D. N.D. N.D. N.D. 6.48 5.30 5.30 5.30 5.30 10 6.12 5.82 6.27 8.945.62 6.18 N.D. N.D. N.D. N.D. N.D. 5.87 5.30 5.30 5.30 5.30 11 7.03 5.6 6.05 7.38 5.60 6.00 N.D. N.D. N.D. N.D. N.D. 6.20 5.30 5.30 5.30 5.30 176.68 7.17 7.08 6.52 6.51 7.07 6.33 5.92 6.61 5.88 6.74 6.50 5.30 5.305.30 5.32 19 6.90 7.35 7.47 6.74 6.58 7.07 7.04 6.29 6.69 5.54 6.55 6.425.30 5.30 5.30 6.04 20 7.01 7.22 7.72 7.31 6.96 7.39 6.73 5.85 6.35 6.735.93 6.37 5.30 5.30 5.37 5.94 21 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 22 6.80 6.98 7.34 7.05 6.43 7.156.22 5.72 6.29 6.56 5.99 6.39 5.30 5.30 5.41 5.98 23 N.D. N.D. N.D. N.D.N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 25 6.66 6.677.07 6.21 5.95 6.79 6.43 6.43 5.93 5.82 5.99 5.35 5.30 5.30 5.39 5.62 26N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.N.D. N.D. 27 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.N.D. N.D. N.D. N.D.

Experimental Discussion

In order to isolate new NPY receptor subtypes an expression cloningapproach was chosen where a functional receptor is actually detectedwith exquisite sensitivity on the surface of transfected cells, using ahighly specific iodinated ligand. Using this strategy, a rathypothalamic cDNA encoding a novel Y-type receptor (Y5) was identified.The fact that 3.5×10⁶ independent clones with a 2.7 kb average insertsize had to be screened to find two clones reveals either a very strongbias against Y5 cDNA cloning in the cDNA library construction procedureor that the Y5 mRNA is expressed at very low levels in rat hypothalamictissue. The longest reading frame in the rat Y5 cDNA (CG-18) encodes a456 amino acid protein with an estimated molecular weight of 50.1 kD.Given there are two N-linked glycosylation sites in the amino terminus,the apparent molecular weight could be slightly higher. The human Y5homolog was isolated from a human hippocampal cDNA library. The longestreading frame in the human Y5 cDNA (CG-19) encodes a 455 amino acidprotein with an estimated molecular weight of 50 kD. The human Y5receptor is one amino acid shorter than the rat Y5 and shows significantamino acid differences both in the N-terminal and the middle of thethird intracellular loop portions of the protein. The seventransmembrane domains and the extracellular loops, however, arevirtually identical and the protein motifs found in both specieshomologs are identical. Both human and rat Y5 receptors carry a largenumber of potential phosphorylation sites in their second and thirdintra-cellular loops which could be involved in the regulation of theirfunctional characteristics.

The rat and human Y5 receptors both carry a leucine zipper in the firstputative transmembrane domain. In such a structure, it has been proposedthat segments containing periodic arrays of leucine residues exist in analpha-helical conformation. The leucine side chains extending from onealpha-helix interact with those from a similar alpha helix of a secondpolypeptide, facilitating dimerization by the formation of a coiled coil(O'Shea et al, 1989). Usually, such patterns are associated with nuclearDNA binding protein like c-myc, c-fos and c-jun, but it is possible thatin some proteins the leucine repeat simply facilitates dimerization andhas little to do with positioning a DNA-binding region. Further evidencesupporting the idea that dimerization of specific seven transmembranereceptors can occur comes from coexpression studies withmuscarinic/adrenergic receptors where intermolecular “cross-talk”between chimeric G-protein coupled receptors has been described (Maggioet al., 1993). The tyrosine phosphorylation site found in the middle ofthis leucine zipper in transmembrane domain on e (TM I) could beinvolved in regulating dimerization of the Y5 receptor. Thephysiological significance of G-protein coupled receptor dimerizationremains to be elucidated, but by analogy with peptide hormone receptorsoligomerization, it could be involved in receptor activation and signaltransduction (Wells, 1994).

The nucleotide and amino acid sequence analysis of Y5 (rat and human)reveals low identity levels with all 7 TM receptors including the Y1, Y2and Y4 receptors, even in the transmembrane domains which are usuallyhighly conserved within receptor subfamilies. CG-18 and CG-19 are namedo receptors because of their unique amino acid sequence (87.2% identicalwith each other, ≦42% identical with the TM regions of previously cloned“Y” receptor subtypes) and pharmacological profile. The name is notbiased toward any one member of the pancreatic polypeptide family.Indeed, the ability of the human Y5 receptor to bind all three knownmembers of the pancreatic polypeptide family (human NPY, human PYY andhuman PP) with similar affinity (Table 8) suggests the concept of a“universal receptor” and provides an argument against using endogenouspeptide ligands for pharmacological classification. The “Y” has itsroots in the original classification of Y1 and Y2 receptor subtypes(Wahlestedt et al., 1987). The letter reflects the conservation inpancreatic polypeptide family members of the C-terminal tyrosine,described as “Y” in the single letter amino acid code. The number is thenext available in the Y-type series, position number three having beenreserved for the pharmacologically defined Y3 receptor. It is noted thatthe cloned human Y1 receptor was introduced by Larhammar and co-workersas a “human neuropeptide Y/peptide YY receptor of the Y1 type”(Larhammar et al., 1992). Similarly, the novel clones described hereincan be described as rat, human and canine neuropeptide Y/peptide YYreceptors of the Y5 type.

An electronic search of the GenBank database for sequences withsimilarity to the human Y5 receptor sequences identified a match betweenthe reverse complement of the human Y5 coding sequence and the human Y1receptor exon IC and its flanking sequences. Exon 1C is located in the5′-untranslated region of the Y1C alternate splice variant mRNA of thehuman Y1 receptor (Ball, et al., 1995). This data reveals that the humanY1 and Y5 receptor genes map, in opposite orientation, to the same locuson chromosome 4q (see FIG. 25).

In addition, a restriction site polymorphism has been described in theY1 receptor gene (Herzog, et al., 1993), 3.1 kb upstream (5′) of the Y1coding sequence and therefore about 21 kb upstream of the Y5 codingregion. It was speculated that this polymorphism in the Y1 receptor geneis associated with changes in feeding behavior because subjectshomozygous for this allele demonstrate a modified feeding behavior,resulting in small changes in energy intake and macronutrient selection(Cote, et al., 1995). However, the observation that the Y1 and Y5receptor genes are co-localized on the same locus and that the efficacyof peptides in in vivo feeding correlates to their in vitro functionalactivity at the Y5 receptor, suggests that this polymorphism isassociated with the Y5 rather than the Y1 gene as was previouslyspeculated. It will be important to characterize the association of thislocus with feeding disorders or obesity in human populations.

The rat hypothalamic Y5 receptor displays a very similar pharmacologicalprofile to the pharmacologically described “atypical” Y1 receptorthought to mediate NPY-induced food intake in rat hypothalamus. Both theY5 receptor and the “feeding receptor” display a preference for NPY andPYY-like analogs, a sensitivity to N-terminal peptide deletion, and atolerance for Pro³⁴. Each would be considered Y1-like except for theanomalous ability of NPY₂ ₃₆ to bind and activate as well as NPY. Eachappears to be sensitive to changes in the mid-region of the peptideligand. For example, a study by Kalra and colleagues (1991) indicatedthat replacement of the NPY midregion by an amino-octanoic chain toproduce NPY₁₋₄-Aca-₂₅₋₃₆ dramatically reduced activity in a feedingbehavioral assay. Likewise, it is noted that the robust difference inhuman PP binding (K_(i)=5.0 nM) and rat PP binding (K_(i)=230) to therat Y5 receptor can be attributed to a series of 8 amino acid changesbetween residues 6-30 in the peptide ligands, with human PP bearing thecloser resemblance to human NPY. Further examination of PP ligandsindicates that those which are capable of activating the Y5 receptorwith high potency, such as bovine and human PP, contain a proline inposition 13 or 14. While this proline is conserved in several PP ligands(porcine, sheep, and canine, for example) and also in human and porcineNPY as well as human and porcine PYY, it is not conserved in rat PP.This structural difference may lead to changes in protein folding andultimately to changes in receptor interaction which underlie therelatively poor potency of rat PP for Y5 receptor activation. Theunderstanding of these structure-activity relationships may be importantfor the design of Y5 selective ligands with the ability to modulate foodintake in vivo.

Noted also that FLRFamide, a structural analog of the FMRFamide peptidewhich is reported to stimulate feeding in rats, was able to bind andactivate the rat Y5 receptor albeit at relatively high concentrations(Orosco, et al., 1989). These matching profiles, combined with aselective activation of the rat Y5 by the reported feeding “modulator”[D-Trp³²]NPY, support the identity of the rat Y5 as the “feedingreceptor” first proposed to explain NPY-induced feeding in rathypothalamus. That the human Y5 receptor has a pharmacological profilelike that of the rat Y5 in both binding and functional assays suggeststhat the two receptors may have similar functions in vivo.

The distribution of Y5 mRNA in rat brain further extends the argumentfor a role of Y5 receptors in feeding behavior. The anatomical locus ofthe feeding response, for example, has been suggested to reside at leastin part in the paraventricular hypothalamic nucleus (PVN) and also inthe lateral hypothalamus, two places where Y5 mRNA was detected inabundance. Post-synaptic localization of the Y5 receptor in both ofthese regions can regulate the response to endogenously released NPY invivo. The paraventricular nucleus receives projections fromNPY-containing neurons in the arcuate nucleus, another region where Y5mRNA was detected. This indicates a pre-synaptic role for the Y5receptor in the control of NPY release via the arcuato-paraventricularprojection, and consequently in the control of feeding behavior. Thelocalization of the Y5 mRNA in the midline thalamic nuclei is alsoimportant. The paraventricular thalamic nucleus/centromedial nucleuscomplex projects heavily to the paraventricular hypothalamus and to theamygdala. As such, the Y5 receptor is a substrate for the emotionalaspect of appetitive behaviors.

Y5 receptors are highly attractive targets for appetite and weightcontrol based on several lines of research (Sahu and Kalra, 1993). NPYis the most potent stimulant of feeding behavior yet described (Clark etal., 1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984). Directinjection of NPY into the hypothalamus of rats can increase food intake˜10-fold over a 4-hour period (Stanley et al., 1992). NPY-stimulatedrats display a preference for carbohydrates over protein and fat(Stanley et al., 1985). Interestingly, NPY and NPY mRNA are increased infood-deprived rats (Brady et al., 1990; O'Shea and Gundlach, 1991) andalso in rats which are genetically obese (Sanacora et al., 1990) or madediabetic by treatment with streptozotocin (White et al., 1990). Onepotential explanation is that NPY, a potent stimulant of feedingbehavior in normal rats, is disregulated in the overweight or diabeticanimal so that food intake is increased, accompanied by obesity. Thephysiological stress of obesity increases the risk for health problemssuch as cardiovascular malfunction, osteoarthritis, andhyperinsulinemia, together with a worsened prognosis for adult-onsetdiabetes. A nonpeptide antagonist targeted to the Y5 receptor couldtherefore be effective as a way to control not only appetite and bodyweight but an entire range of obesity- and diabetes-related disorders(Dryden et al., 1994). There is also neurochemical evidence to suggestthat NPY-mediated functions are disregulated in eating disorders such asbulimia and anorexia nervosa, so that they too could be responsive totreatment by a Y5-selective drug. It has been proposed, for example,that food intake in NPY-stimulated rats mimics the massive foodconsumption associated with binge eating in bulimia (Stanley, 1993).Cerebro-spinal fluid (CSF) levels of PYY but not NPY were elevated inbulimic patients who abstained from binging, and then diminished whenbinging was allowed (Berrettini et al., 1988). Conversely, NPY levelswere elevated in underweight anorectic patients and then diminished asbody weight was normalized (Kaye et al., 1990).

As described above, the human and rat in vitro expression models wereused in combination to screen for compounds intended to modulateNPY-dependent feeding behavior. Using this approach, several compoundswere discovered which inhibit feeding behavior in animal models, whichshould lead to additional drug discoveries.

The characterization of the canine Y5 receptor in porcine ¹²⁵I-PYYbinding assays with human analogs of NPY, PYY and PP provides a logicalbasis for comparison with the human and rat receptor homologs. Thepeptides also have relevance in the context of canine physiology. NPY ishighly conserved across species (e.g. 100% in human, rat, guinea pig,rabbit and alligator) such that canine NPY is predicted to resemblehuman NPY, although the sequence of canine NPY is currently unknown.Canine and human PYY differ in only 2 out of 36 positions, whereascanine PYY is identical to porcine PYY. Finally, human and canine PPdeviate in only 2 out of 36 residues. Thus, the canine Y5 receptorappears to be a plausible target not only for NPY synthesized in thecanine nervous system, but also for circulating or neurally-derived PYYand pp. Given the general conservation in structure and pharmacology ofY5 receptors, it is hypothesized that the canine Y5 receptor mediatesall of the functions proposed for human and rat Y5 receptors, includingthe stimulation of feeding behavior. The cloned canine Y5 receptor andcanine in vivo models are therefore believed to comprise a useful systemwith which to evaluate biological actions of Y5-selective compounds forthe treatment of obesity and eating disorders in humans.

The Y5 pharmacological profile further offers a new standard by which toreview the molecular basis of all NPY-dependent processes; examples arelisted in Table 18. Such an exercise suggests that the Y5 receptor islikely to have a physiological significance beyond feeding behavior. Ithas been reported, for example, that a Y-type receptor can regulateluteinizing hormone releasing hormone (LHRH) release from the medianeminence of steroid-primed rats in vitro with an atypical Y1pharmacological profile. NPY, NPY₂₋₃₆, and LP-NPY were all effective at1 uM but deletion of as few as four amino acids from the N-terminus ofNPY destroyed biological activity. The Y5 may therefore represent atherapeutic target for sexual or reproductive disorders. Preliminary insitu hybridization of rat Y5 mRNA in hippocampus and elsewhere furthersuggest that additional roles will be uncovered, for example, in theregulation of memory. The localization of Y5 mRNA in amygdala alsosuggests a potential role for Y5 receptor modulation in affectivedisorders such as depression and anxiety. It is worth while consideringthat the Y5 is so similar in pharmacological profile to the other Y-typereceptors that it may have been overlooked among a mixed population ofY1, Y2 and Y4 receptors. Certain functions now associated with thesesubtypes could therefore be reassigned to Y5 as pharmacological toolsgrow more sophisticated (Table 18). By offering new insight into NPYreceptor pharmacology, the Y5 thereby provides a greater clarity andfocus in the field of drug design.

TABLE 18 Papthophysiological Conditions Associated With NPY Thefollowing pathological conditions have been linked to either 1)application of exogenous NPY, or 2) changes in levels of endogenous NPY. 1 obesity Sahu and Kalra, 1993  2 eating disorders Stanley, 1993(anorexia and bulimia nervosa)  3 sexual/reproductive Clark, 1994function  4 depression Heilig and Weiderlov, 1990  5 anxiety Wahlestedtet al., 1993  6 cocaine Wahlestedt et al., 1991 addiction  7 gastriculcer Penner et al., 1993  8 memory loss Morley and Flood, 1990  9 painHua et al., 1991 10 epileptic seizure Rizzi et al., 1993 11 hypertensionZukowska-Grojec et al., 1993 12 subarachnoid Abel et al., 1988hemorrhage 13 shock Hauser et al., 1993 14 circadian rhythm Albers andFerris, 1984 15 nasal congestion Lacroix et al., 1988 16 diarrhea Coxand Cuthbert, 1990 17 neurogenic Zoubek et al., 1993 voiding dysfunction

A successful strategy for the design of a Y5-receptor based drug or forany drug targeted to single G protein-coupled receptor subtype involvesthe screening of candidate compounds 1) in radioligand binding assays soas to detect affinity for cross-reactive G protein-coupled receptors,and 2) in physiological assays so as to detect undesirable side effects.In the specific process of screening for a Y5-selective drug, thereceptor subtypes most likely to cross-react and therefore mostimportant for radioligand binding screens include the other “Y-type”receptors, Y1, Y2, Y3, and Y4. Cross-reactivity between the Y5 and anyof the other subtypes could a result in potential complications assuggested by the pathophysiological indications listed in Table 18. Indesigning a Y5 antagonist for obesity and appetite control, for example,it is important not to design a Y1 antagonist resulting in hypertensionor increased anxiety, a Y2 antagonist resulting in memory loss, or a Y4antagonist resulting in increased appetite.

TABLE 19 Y-Type Receptor Indications Y-type Receptor Receptor DrugIndications Subtype Activity Reference obesity, atypical Y1 antagonistSahu and appetite Kalra, disorder 1993 adult onset atypical Y1antagonist Sahu and diabetes Kalra, 1993 bulimia atypical Y1 antagonistStanley, nervosa 1993 pheochromocytoma- Y1 antagonist Grouzman inducedet al., hypertension 1989 subarachnoid Y1 antagonist Abel et al.,hemorrhage 1988 neurogenic Y1 antagonist Zukowska- vascular Y2antagonist Grojec et al., hypertrophy 1993 epileptic Y2 antagonist Rizziet al., seizure 1993 hypertension: peripheral Y1 antagonist Grundemarcentral, central Y3 agonist and peripheral central Y2 antagonistHakanson, regulation 1993 Barraco et al., 1991 obesity, Y4 or PP agonistMalaisse- appetite Lagae et al., disorder 1977 anorexia atypical Y1agonist Berrettini nervosa et al., 1988 anxiety Y1 agonist Wahlestedt etal., 1993 cocaine Y1 agonist Wahlestedt addiction et al., stress- Y1agonist Penner et al., induced Y4 or PP agonist 1993 gastric ulcermemory loss Y2 agonist Morley and Flood, 1990 pain Y2 agonist Hua etal., 1991 shock Y1 agonist Hauser et al., 1993 sleep Y2 not clear Albersdisturbances, and jet lag Ferris, 1984 nasal Y1 agonist Lacroixdecongestion Y2 agonist et al., 1988 diarrhea Y2 agonist Cox andCuthbert, 1990

The cloning of the Y5 receptor from human and rat is especially valuablefor receptor characterization based on in situ localization, anti-sensefunctional knock-out, and gene induction. These studies will generateimportant information related to Y5 receptor function and itstherapeutic significance. The cloned Y5 receptor lends itself tomutagenesis studies in which receptor/ligand interactions can bemodeled. The Y5 receptor further allows us to investigate thepossibility of other Y-type receptors through homology cloning. Thesecould include new receptor subtypes as well as Y5 species homologs forthe establishment of experimental animal models with relevance for humanpathology. The Y5 receptor therefore represents an enormous opportunityfor the development of novel and selective drug therapies, particularlythose targeted to appetite and weight control, but also for memory loss,depression, anxiety, gastric ulcer, epileptic seizure, pain,hypertension, subarachnoid hemorrhage, sleeping disturbances, nasalcongestion, neurogenic voiding dysfuncion, and diarrhea.

In particular, the discovery of Y5-selective antagonists which inhibitfood intake in rats provides a method of modifying feeding behavior in awide variety of vertebrate animals.

TABLE 20 Pharmacological profile of the canine Y5 receptor. IC₅₀ valuesfrom competitive displacement of porcine ¹²⁵I-PYY binding to membranesof COS-7 cells transiently transfected with canine Y5 receptor cDNA wereconverted to K_(i) values according to the Cheng-Prusoff equation, K_(i)= IC₅₀/(1 + [L]/K_(d)). For all peptides, n = 2. For BIBP 3226, n = 3.Peptide K_(i) NPY, human 2.2 NPY, porcine 6.2 NPY₂₋₃₆, porcine 2.1NPY₃₋₃₆, porcine 16 NPY₁₃₋₃₆, porcine 120 [Leu³¹, Pro³⁴]NPY, 4.1 porcineC2-NPY, porcine 300 D-[Trp³²]NPY, human 35 PYY, human 3.2 PYY₃₋₃₆, human14 [Pro³⁴]PYY, human 1.4 PP, human 6.3 PP, bovine 10 PP, rat 160 BIBP3226 17000

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24 1 1501 DNA Rattus norvegicus CDS (61)..(1431) 1 ttagttttgt tctgagaacgttagagttat agtaccgtgc gatcgttctt caagctgcta 60 atg gac gtc ctc ttc ttccac cag gat tct agt atg gag ttt aag ctt 108 Met Asp Val Leu Phe Phe HisGln Asp Ser Ser Met Glu Phe Lys Leu 1 5 10 15 gag gag cat ttt aac aagaca ttt gtc aca gag aac aat aca gct gct 156 Glu Glu His Phe Asn Lys ThrPhe Val Thr Glu Asn Asn Thr Ala Ala 20 25 30 gct cgg aat gca gcc ttc cctgcc tgg gag gac tac aga ggc agc gta 204 Ala Arg Asn Ala Ala Phe Pro AlaTrp Glu Asp Tyr Arg Gly Ser Val 35 40 45 gac gat tta caa tac ttt ctg attggg ctc tat aca ttc gta agt ctt 252 Asp Asp Leu Gln Tyr Phe Leu Ile GlyLeu Tyr Thr Phe Val Ser Leu 50 55 60 ctt ggc ttt atg ggc aat cta ctt atttta atg gct gtt atg aaa aag 300 Leu Gly Phe Met Gly Asn Leu Leu Ile LeuMet Ala Val Met Lys Lys 65 70 75 80 cgc aat cag aag act aca gtg aac tttctc ata ggc aac ctg gcc ttc 348 Arg Asn Gln Lys Thr Thr Val Asn Phe LeuIle Gly Asn Leu Ala Phe 85 90 95 tcc gac atc ttg gtc gtc ctg ttt tgc tcccct ttc acc ctg acc tct 396 Ser Asp Ile Leu Val Val Leu Phe Cys Ser ProPhe Thr Leu Thr Ser 100 105 110 gtc ttg ttg gat cag tgg atg ttt ggc aaagcc atg tgc cat atc atg 444 Val Leu Leu Asp Gln Trp Met Phe Gly Lys AlaMet Cys His Ile Met 115 120 125 ccg ttc ctt caa tgt gtg tca gtt ctg gtttca act ctg att tta ata 492 Pro Phe Leu Gln Cys Val Ser Val Leu Val SerThr Leu Ile Leu Ile 130 135 140 tca att gcc att gtc agg tat cat atg ataaag cac cct att tct aac 540 Ser Ile Ala Ile Val Arg Tyr His Met Ile LysHis Pro Ile Ser Asn 145 150 155 160 aat tta acg gca aac cat ggc tac ttcctg ata gct act gtc tgg aca 588 Asn Leu Thr Ala Asn His Gly Tyr Phe LeuIle Ala Thr Val Trp Thr 165 170 175 ctg ggc ttt gcc atc tgt tct ccc ctccca gtg ttt cac agt ctt gtg 636 Leu Gly Phe Ala Ile Cys Ser Pro Leu ProVal Phe His Ser Leu Val 180 185 190 gaa ctt aag gag acc ttt ggc tca gcactg ctg agt agc aaa tat ctc 684 Glu Leu Lys Glu Thr Phe Gly Ser Ala LeuLeu Ser Ser Lys Tyr Leu 195 200 205 tgt gtt gag tca tgg ccc tct gat tcatac aga att gct ttc aca atc 732 Cys Val Glu Ser Trp Pro Ser Asp Ser TyrArg Ile Ala Phe Thr Ile 210 215 220 tct tta ttg cta gtg cag tat atc ctgcct cta gta tgt tta acg gta 780 Ser Leu Leu Leu Val Gln Tyr Ile Leu ProLeu Val Cys Leu Thr Val 225 230 235 240 agt cat acc agc gtc tgc cga agcata agc tgt gga ttg tcc cac aaa 828 Ser His Thr Ser Val Cys Arg Ser IleSer Cys Gly Leu Ser His Lys 245 250 255 gaa aac aga ctc gaa gaa aat gagatg atc aac tta acc cta cag cca 876 Glu Asn Arg Leu Glu Glu Asn Glu MetIle Asn Leu Thr Leu Gln Pro 260 265 270 tcc aaa aag agc agg aac cag gcaaaa acc ccc agc act caa aag tgg 924 Ser Lys Lys Ser Arg Asn Gln Ala LysThr Pro Ser Thr Gln Lys Trp 275 280 285 agc tac tca ttc atc aga aag cacaga agg agg tac agc aag aag acg 972 Ser Tyr Ser Phe Ile Arg Lys His ArgArg Arg Tyr Ser Lys Lys Thr 290 295 300 gcc tgt gtc tta ccc gcc cca gcagga cct tcc cag ggg aag cac cta 1020 Ala Cys Val Leu Pro Ala Pro Ala GlyPro Ser Gln Gly Lys His Leu 305 310 315 320 gcc gtt cca gaa aat cca gcctcc gtc cgt agc cag ctg tcg cca tcc 1068 Ala Val Pro Glu Asn Pro Ala SerVal Arg Ser Gln Leu Ser Pro Ser 325 330 335 agt aag gtc att cca ggg gtccca atc tgc ttt gag gtg aaa cct gaa 1116 Ser Lys Val Ile Pro Gly Val ProIle Cys Phe Glu Val Lys Pro Glu 340 345 350 gaa agc tca gat gct cat gagatg aga gtc aag cgt tcc atc act aga 1164 Glu Ser Ser Asp Ala His Glu MetArg Val Lys Arg Ser Ile Thr Arg 355 360 365 ata aaa aag aga tct cga agtgtt ttc tac aga ctg acc ata ctg ata 1212 Ile Lys Lys Arg Ser Arg Ser ValPhe Tyr Arg Leu Thr Ile Leu Ile 370 375 380 ctc gtg ttc gcc gtt agc tggatg cca ctc cac gtc ttc cac gtg gtg 1260 Leu Val Phe Ala Val Ser Trp MetPro Leu His Val Phe His Val Val 385 390 395 400 act gac ttc aat gat aacttg att tcc aat agg cat ttc aag ctg gta 1308 Thr Asp Phe Asn Asp Asn LeuIle Ser Asn Arg His Phe Lys Leu Val 405 410 415 tac tgc atc tgt cac ttgtta ggc atg atg tcc tgt tgt cta aat ccg 1356 Tyr Cys Ile Cys His Leu LeuGly Met Met Ser Cys Cys Leu Asn Pro 420 425 430 atc cta tat ggt ttc cttaat aat ggt atc aaa gca gac ttg aga gcc 1404 Ile Leu Tyr Gly Phe Leu AsnAsn Gly Ile Lys Ala Asp Leu Arg Ala 435 440 445 ctt atc cac tgc cta cacatg tca tga ttctctctgt gcaccaaaga 1451 Leu Ile His Cys Leu His Met Ser450 455 gagaagaaac gtggtaattg acacataatt tatacagaag tattctggat 1501 2456 PRT Rattus norvegicus 2 Met Asp Val Leu Phe Phe His Gln Asp Ser SerMet Glu Phe Lys Leu 1 5 10 15 Glu Glu His Phe Asn Lys Thr Phe Val ThrGlu Asn Asn Thr Ala Ala 20 25 30 Ala Arg Asn Ala Ala Phe Pro Ala Trp GluAsp Tyr Arg Gly Ser Val 35 40 45 Asp Asp Leu Gln Tyr Phe Leu Ile Gly LeuTyr Thr Phe Val Ser Leu 50 55 60 Leu Gly Phe Met Gly Asn Leu Leu Ile LeuMet Ala Val Met Lys Lys 65 70 75 80 Arg Asn Gln Lys Thr Thr Val Asn PheLeu Ile Gly Asn Leu Ala Phe 85 90 95 Ser Asp Ile Leu Val Val Leu Phe CysSer Pro Phe Thr Leu Thr Ser 100 105 110 Val Leu Leu Asp Gln Trp Met PheGly Lys Ala Met Cys His Ile Met 115 120 125 Pro Phe Leu Gln Cys Val SerVal Leu Val Ser Thr Leu Ile Leu Ile 130 135 140 Ser Ile Ala Ile Val ArgTyr His Met Ile Lys His Pro Ile Ser Asn 145 150 155 160 Asn Leu Thr AlaAsn His Gly Tyr Phe Leu Ile Ala Thr Val Trp Thr 165 170 175 Leu Gly PheAla Ile Cys Ser Pro Leu Pro Val Phe His Ser Leu Val 180 185 190 Glu LeuLys Glu Thr Phe Gly Ser Ala Leu Leu Ser Ser Lys Tyr Leu 195 200 205 CysVal Glu Ser Trp Pro Ser Asp Ser Tyr Arg Ile Ala Phe Thr Ile 210 215 220Ser Leu Leu Leu Val Gln Tyr Ile Leu Pro Leu Val Cys Leu Thr Val 225 230235 240 Ser His Thr Ser Val Cys Arg Ser Ile Ser Cys Gly Leu Ser His Lys245 250 255 Glu Asn Arg Leu Glu Glu Asn Glu Met Ile Asn Leu Thr Leu GlnPro 260 265 270 Ser Lys Lys Ser Arg Asn Gln Ala Lys Thr Pro Ser Thr GlnLys Trp 275 280 285 Ser Tyr Ser Phe Ile Arg Lys His Arg Arg Arg Tyr SerLys Lys Thr 290 295 300 Ala Cys Val Leu Pro Ala Pro Ala Gly Pro Ser GlnGly Lys His Leu 305 310 315 320 Ala Val Pro Glu Asn Pro Ala Ser Val ArgSer Gln Leu Ser Pro Ser 325 330 335 Ser Lys Val Ile Pro Gly Val Pro IleCys Phe Glu Val Lys Pro Glu 340 345 350 Glu Ser Ser Asp Ala His Glu MetArg Val Lys Arg Ser Ile Thr Arg 355 360 365 Ile Lys Lys Arg Ser Arg SerVal Phe Tyr Arg Leu Thr Ile Leu Ile 370 375 380 Leu Val Phe Ala Val SerTrp Met Pro Leu His Val Phe His Val Val 385 390 395 400 Thr Asp Phe AsnAsp Asn Leu Ile Ser Asn Arg His Phe Lys Leu Val 405 410 415 Tyr Cys IleCys His Leu Leu Gly Met Met Ser Cys Cys Leu Asn Pro 420 425 430 Ile LeuTyr Gly Phe Leu Asn Asn Gly Ile Lys Ala Asp Leu Arg Ala 435 440 445 LeuIle His Cys Leu His Met Ser 450 455 3 1457 DNA Homo sapiens CDS(61)..(1428) 3 gtttccctct gaatagatta atttaaagta gtcatgtaat gtttttttggttgctgacaa 60 atg tct ttt tat tcc aag cag gac tat aat atg gat tta gagctc gac 108 Met Ser Phe Tyr Ser Lys Gln Asp Tyr Asn Met Asp Leu Glu LeuAsp 1 5 10 15 gag tat tat aac aag aca ctt gcc aca gag aat aat act gctgcc act 156 Glu Tyr Tyr Asn Lys Thr Leu Ala Thr Glu Asn Asn Thr Ala AlaThr 20 25 30 cgg aat tct gat ttc cca gtc tgg gat gac tat aaa agc agt gtagat 204 Arg Asn Ser Asp Phe Pro Val Trp Asp Asp Tyr Lys Ser Ser Val Asp35 40 45 gac tta cag tat ttt ctg att ggg ctc tat aca ttt gta agt ctt ctt252 Asp Leu Gln Tyr Phe Leu Ile Gly Leu Tyr Thr Phe Val Ser Leu Leu 5055 60 ggc ttt atg ggg aat cta ctt att tta atg gct ctc atg aaa aag cgt300 Gly Phe Met Gly Asn Leu Leu Ile Leu Met Ala Leu Met Lys Lys Arg 6570 75 80 aat cag aag act acg gta aac ttc ctc ata ggc aat ctg gcc ttt tct348 Asn Gln Lys Thr Thr Val Asn Phe Leu Ile Gly Asn Leu Ala Phe Ser 8590 95 gat atc ttg gtt gtg ctg ttt tgc tca cct ttc aca ctg acg tct gtc396 Asp Ile Leu Val Val Leu Phe Cys Ser Pro Phe Thr Leu Thr Ser Val 100105 110 ttg ctg gat cag tgg atg ttt ggc aaa gtc atg tgc cat att atg cct444 Leu Leu Asp Gln Trp Met Phe Gly Lys Val Met Cys His Ile Met Pro 115120 125 ttt ctt caa tgt gtg tca gtt ttg gtt tca act tta att tta ata tca492 Phe Leu Gln Cys Val Ser Val Leu Val Ser Thr Leu Ile Leu Ile Ser 130135 140 att gcc att gtc agg tat cat atg ata aaa cat ccc ata tct aat aat540 Ile Ala Ile Val Arg Tyr His Met Ile Lys His Pro Ile Ser Asn Asn 145150 155 160 tta aca gca aac cat ggc tac ttt ctg ata gct act gtc tgg acacta 588 Leu Thr Ala Asn His Gly Tyr Phe Leu Ile Ala Thr Val Trp Thr Leu165 170 175 ggt ttt gcc atc tgt tct ccc ctt cca gtg ttt cac agt ctt gtggaa 636 Gly Phe Ala Ile Cys Ser Pro Leu Pro Val Phe His Ser Leu Val Glu180 185 190 ctt caa gaa aca ttt ggt tca gca ttg ctg agc agc agg tat ttatgt 684 Leu Gln Glu Thr Phe Gly Ser Ala Leu Leu Ser Ser Arg Tyr Leu Cys195 200 205 gtt gag tca tgg cca tct gat tca tac aga att gcc ttt act atctct 732 Val Glu Ser Trp Pro Ser Asp Ser Tyr Arg Ile Ala Phe Thr Ile Ser210 215 220 tta ttg cta gtt cag tat att ctg ccc tta gtt tgt ctt act gtaagt 780 Leu Leu Leu Val Gln Tyr Ile Leu Pro Leu Val Cys Leu Thr Val Ser225 230 235 240 cat aca agt gtc tgc aga agt ata agc tgt gga ttg tcc aacaaa gaa 828 His Thr Ser Val Cys Arg Ser Ile Ser Cys Gly Leu Ser Asn LysGlu 245 250 255 aac aga ctt gaa gaa aat gag atg atc aac tta act ctt catcca tcc 876 Asn Arg Leu Glu Glu Asn Glu Met Ile Asn Leu Thr Leu His ProSer 260 265 270 aaa aag agt ggg cct cag gtg aaa ctc tct ggc agc cat aaatgg agt 924 Lys Lys Ser Gly Pro Gln Val Lys Leu Ser Gly Ser His Lys TrpSer 275 280 285 tat tca ttc atc aaa aaa cac aga aga aga tat agc aag aagaca gca 972 Tyr Ser Phe Ile Lys Lys His Arg Arg Arg Tyr Ser Lys Lys ThrAla 290 295 300 tgt gtg tta cct gct cca gaa aga cct tct caa gag aac cactcc aga 1020 Cys Val Leu Pro Ala Pro Glu Arg Pro Ser Gln Glu Asn His SerArg 305 310 315 320 ata ctt cca gaa aac ttt ggc tct gta aga agt cag ctctct tca tcc 1068 Ile Leu Pro Glu Asn Phe Gly Ser Val Arg Ser Gln Leu SerSer Ser 325 330 335 agt aag ttc ata cca ggg gtc ccc act tgc ttt gag ataaaa cct gaa 1116 Ser Lys Phe Ile Pro Gly Val Pro Thr Cys Phe Glu Ile LysPro Glu 340 345 350 gaa aat tca gat gtt cat gaa ttg aga gta aaa cgt tctgtt aca aga 1164 Glu Asn Ser Asp Val His Glu Leu Arg Val Lys Arg Ser ValThr Arg 355 360 365 ata aaa aag aga tct cga agt gtt ttc tac aga ctg accata ctg ata 1212 Ile Lys Lys Arg Ser Arg Ser Val Phe Tyr Arg Leu Thr IleLeu Ile 370 375 380 tta gta ttt gct gtt agt tgg atg cca cta cac ctt ttccat gtg gta 1260 Leu Val Phe Ala Val Ser Trp Met Pro Leu His Leu Phe HisVal Val 385 390 395 400 act gat ttt aat gac aat ctt att tca aat agg catttc aag ttg gtg 1308 Thr Asp Phe Asn Asp Asn Leu Ile Ser Asn Arg His PheLys Leu Val 405 410 415 tat tgc att tgt cat ttg ttg ggc atg atg tcc tgttgt ctt aat cca 1356 Tyr Cys Ile Cys His Leu Leu Gly Met Met Ser Cys CysLeu Asn Pro 420 425 430 att cta tat ggg ttt ctt aat aat ggg att aaa gctgat tta gtg tcc 1404 Ile Leu Tyr Gly Phe Leu Asn Asn Gly Ile Lys Ala AspLeu Val Ser 435 440 445 ctt ata cac tgt ctt cat atg taa taattctcactgtttaccaa ggaaagaac 1457 Leu Ile His Cys Leu His Met 450 455 4 455 PRTHomo sapiens 4 Met Ser Phe Tyr Ser Lys Gln Asp Tyr Asn Met Asp Leu GluLeu Asp 1 5 10 15 Glu Tyr Tyr Asn Lys Thr Leu Ala Thr Glu Asn Asn ThrAla Ala Thr 20 25 30 Arg Asn Ser Asp Phe Pro Val Trp Asp Asp Tyr Lys SerSer Val Asp 35 40 45 Asp Leu Gln Tyr Phe Leu Ile Gly Leu Tyr Thr Phe ValSer Leu Leu 50 55 60 Gly Phe Met Gly Asn Leu Leu Ile Leu Met Ala Leu MetLys Lys Arg 65 70 75 80 Asn Gln Lys Thr Thr Val Asn Phe Leu Ile Gly AsnLeu Ala Phe Ser 85 90 95 Asp Ile Leu Val Val Leu Phe Cys Ser Pro Phe ThrLeu Thr Ser Val 100 105 110 Leu Leu Asp Gln Trp Met Phe Gly Lys Val MetCys His Ile Met Pro 115 120 125 Phe Leu Gln Cys Val Ser Val Leu Val SerThr Leu Ile Leu Ile Ser 130 135 140 Ile Ala Ile Val Arg Tyr His Met IleLys His Pro Ile Ser Asn Asn 145 150 155 160 Leu Thr Ala Asn His Gly TyrPhe Leu Ile Ala Thr Val Trp Thr Leu 165 170 175 Gly Phe Ala Ile Cys SerPro Leu Pro Val Phe His Ser Leu Val Glu 180 185 190 Leu Gln Glu Thr PheGly Ser Ala Leu Leu Ser Ser Arg Tyr Leu Cys 195 200 205 Val Glu Ser TrpPro Ser Asp Ser Tyr Arg Ile Ala Phe Thr Ile Ser 210 215 220 Leu Leu LeuVal Gln Tyr Ile Leu Pro Leu Val Cys Leu Thr Val Ser 225 230 235 240 HisThr Ser Val Cys Arg Ser Ile Ser Cys Gly Leu Ser Asn Lys Glu 245 250 255Asn Arg Leu Glu Glu Asn Glu Met Ile Asn Leu Thr Leu His Pro Ser 260 265270 Lys Lys Ser Gly Pro Gln Val Lys Leu Ser Gly Ser His Lys Trp Ser 275280 285 Tyr Ser Phe Ile Lys Lys His Arg Arg Arg Tyr Ser Lys Lys Thr Ala290 295 300 Cys Val Leu Pro Ala Pro Glu Arg Pro Ser Gln Glu Asn His SerArg 305 310 315 320 Ile Leu Pro Glu Asn Phe Gly Ser Val Arg Ser Gln LeuSer Ser Ser 325 330 335 Ser Lys Phe Ile Pro Gly Val Pro Thr Cys Phe GluIle Lys Pro Glu 340 345 350 Glu Asn Ser Asp Val His Glu Leu Arg Val LysArg Ser Val Thr Arg 355 360 365 Ile Lys Lys Arg Ser Arg Ser Val Phe TyrArg Leu Thr Ile Leu Ile 370 375 380 Leu Val Phe Ala Val Ser Trp Met ProLeu His Leu Phe His Val Val 385 390 395 400 Thr Asp Phe Asn Asp Asn LeuIle Ser Asn Arg His Phe Lys Leu Val 405 410 415 Tyr Cys Ile Cys His LeuLeu Gly Met Met Ser Cys Cys Leu Asn Pro 420 425 430 Ile Leu Tyr Gly PheLeu Asn Asn Gly Ile Lys Ala Asp Leu Val Ser 435 440 445 Leu Ile His CysLeu His Met 450 455 5 1054 DNA Canine CDS (3)..(1007) 5 tc atg tgt cacatt atg cct ttt ctt caa tgt gtg tca gtt ctg gtt 47 Met Cys His Ile MetPro Phe Leu Gln Cys Val Ser Val Leu Val 1 5 10 15 tca act tta att ctaata tca att gcc att gtc agg tat cat atg atc 95 Ser Thr Leu Ile Leu IleSer Ile Ala Ile Val Arg Tyr His Met Ile 20 25 30 aag cat cct ata tct aacaat tta aca gca aac cat ggc tac ttc ctg 143 Lys His Pro Ile Ser Asn AsnLeu Thr Ala Asn His Gly Tyr Phe Leu 35 40 45 att gct act gtc tgg aca ctaggt ttt gcg att tgt tct ccc ctt cca 191 Ile Ala Thr Val Trp Thr Leu GlyPhe Ala Ile Cys Ser Pro Leu Pro 50 55 60 gtg ttt cac agt ctg gtg gaa cttcag gaa aca ttt gac tcc gca ttg 239 Val Phe His Ser Leu Val Glu Leu GlnGlu Thr Phe Asp Ser Ala Leu 65 70 75 ctg agc agc agg tat tta tgt gtt gagtcg tgg cca tct gat tcg tac 287 Leu Ser Ser Arg Tyr Leu Cys Val Glu SerTrp Pro Ser Asp Ser Tyr 80 85 90 95 aga atc gct ttt act atc tct tta ttgcta gtc cag tat att ctt ccc 335 Arg Ile Ala Phe Thr Ile Ser Leu Leu LeuVal Gln Tyr Ile Leu Pro 100 105 110 ttg gtg tgt cta act gtg agc cat accagt gtc tgc agg agt ata agc 383 Leu Val Cys Leu Thr Val Ser His Thr SerVal Cys Arg Ser Ile Ser 115 120 125 tgc ggg ttg tcc aac aaa gaa aac aaactg gaa gaa aac gag atg atc 431 Cys Gly Leu Ser Asn Lys Glu Asn Lys LeuGlu Glu Asn Glu Met Ile 130 135 140 aac tta act ctt caa cca ttc aaa aagagt ggg cct cag gtg aaa ctt 479 Asn Leu Thr Leu Gln Pro Phe Lys Lys SerGly Pro Gln Val Lys Leu 145 150 155 tcc agc agc cat aaa tgg agc tat tcattc atc aga aaa cac agg aga 527 Ser Ser Ser His Lys Trp Ser Tyr Ser PheIle Arg Lys His Arg Arg 160 165 170 175 agg tac agc aag aag acg gcg tgtgtc tta cct gct cca gca aga cct 575 Arg Tyr Ser Lys Lys Thr Ala Cys ValLeu Pro Ala Pro Ala Arg Pro 180 185 190 cct caa gag aac cac tca aga atgctt cca gaa aac ttt ggt tct gta 623 Pro Gln Glu Asn His Ser Arg Met LeuPro Glu Asn Phe Gly Ser Val 195 200 205 aga agt cag cat tct tca tcc agtaag ttc ata ccg ggg gtc ccc acc 671 Arg Ser Gln His Ser Ser Ser Ser LysPhe Ile Pro Gly Val Pro Thr 210 215 220 tgc ttt gag gtg aaa cct gaa gaaaac tcg gat gtt cat gac atg aga 719 Cys Phe Glu Val Lys Pro Glu Glu AsnSer Asp Val His Asp Met Arg 225 230 235 gta aac cgt tct atc atg aga atcaaa aag aga tcc cga agt gtt ttc 767 Val Asn Arg Ser Ile Met Arg Ile LysLys Arg Ser Arg Ser Val Phe 240 245 250 255 tat aga cta acc ata ctg atacta gtg ttt gcc gtt agc tgg atg cca 815 Tyr Arg Leu Thr Ile Leu Ile LeuVal Phe Ala Val Ser Trp Met Pro 260 265 270 cta cac ctt ttc cat gtg gtaact gat ttt aat gac aac ctc att tca 863 Leu His Leu Phe His Val Val ThrAsp Phe Asn Asp Asn Leu Ile Ser 275 280 285 aac agg cat ttc aaa ttg gtgtat tgc att tgt cat ttg tta ggc atg 911 Asn Arg His Phe Lys Leu Val TyrCys Ile Cys His Leu Leu Gly Met 290 295 300 atg tcc tgt tgt ctt aat cctatt ctg tat ggt ttt ctc aat aat ggg 959 Met Ser Cys Cys Leu Asn Pro IleLeu Tyr Gly Phe Leu Asn Asn Gly 305 310 315 atc aaa gct gat tta att tccctt ata cag tgt ctt cat atg tca taa 1007 Ile Lys Ala Asp Leu Ile Ser LeuIle Gln Cys Leu His Met Ser 320 325 330 ttattaatgt ttaccaagga gacaacaaatgttgggatcg tctaaaa 1054 6 334 PRT Canine 6 Met Cys His Ile Met Pro PheLeu Gln Cys Val Ser Val Leu Val Ser 1 5 10 15 Thr Leu Ile Leu Ile SerIle Ala Ile Val Arg Tyr His Met Ile Lys 20 25 30 His Pro Ile Ser Asn AsnLeu Thr Ala Asn His Gly Tyr Phe Leu Ile 35 40 45 Ala Thr Val Trp Thr LeuGly Phe Ala Ile Cys Ser Pro Leu Pro Val 50 55 60 Phe His Ser Leu Val GluLeu Gln Glu Thr Phe Asp Ser Ala Leu Leu 65 70 75 80 Ser Ser Arg Tyr LeuCys Val Glu Ser Trp Pro Ser Asp Ser Tyr Arg 85 90 95 Ile Ala Phe Thr IleSer Leu Leu Leu Val Gln Tyr Ile Leu Pro Leu 100 105 110 Val Cys Leu ThrVal Ser His Thr Ser Val Cys Arg Ser Ile Ser Cys 115 120 125 Gly Leu SerAsn Lys Glu Asn Lys Leu Glu Glu Asn Glu Met Ile Asn 130 135 140 Leu ThrLeu Gln Pro Phe Lys Lys Ser Gly Pro Gln Val Lys Leu Ser 145 150 155 160Ser Ser His Lys Trp Ser Tyr Ser Phe Ile Arg Lys His Arg Arg Arg 165 170175 Tyr Ser Lys Lys Thr Ala Cys Val Leu Pro Ala Pro Ala Arg Pro Pro 180185 190 Gln Glu Asn His Ser Arg Met Leu Pro Glu Asn Phe Gly Ser Val Arg195 200 205 Ser Gln His Ser Ser Ser Ser Lys Phe Ile Pro Gly Val Pro ThrCys 210 215 220 Phe Glu Val Lys Pro Glu Glu Asn Ser Asp Val His Asp MetArg Val 225 230 235 240 Asn Arg Ser Ile Met Arg Ile Lys Lys Arg Ser ArgSer Val Phe Tyr 245 250 255 Arg Leu Thr Ile Leu Ile Leu Val Phe Ala ValSer Trp Met Pro Leu 260 265 270 His Leu Phe His Val Val Thr Asp Phe AsnAsp Asn Leu Ile Ser Asn 275 280 285 Arg His Phe Lys Leu Val Tyr Cys IleCys His Leu Leu Gly Met Met 290 295 300 Ser Cys Cys Leu Asn Pro Ile LeuTyr Gly Phe Leu Asn Asn Gly Ile 305 310 315 320 Lys Ala Asp Leu Ile SerLeu Ile Gln Cys Leu His Met Ser 325 330 7 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 7 tggatcagtg gatgtttggc aaag24 8 28 DNA Artificial Sequence Description of Artificial SequencePrimer 8 gtctgtagaa aacacttcga gatctctt 28 9 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 9 cttccagtgt ttcacagtct ggtgg25 10 25 DNA Artificial Sequence Description of Artificial SequencePrimer 10 ctgagcagca ggtatttatg tgttg 25 11 28 DNA Artificial SequenceDescription of Artificial Sequence Primer 11 ctggatgaag aatgctgacttcttacag 28 12 25 DNA Artificial Sequence Description of ArtificialSequence Primer 12 ttcttgagtg gttctcttga ggagg 25 13 1479 DNA Canine CDS(62)..(1432) 13 gtagtctccc tctcagaatt gatttatcgt agtcatgtaa ttttttaaaagttggtaact 60 a atg tct ttt tat tcc aag cag aac tct aag atg gat tta gaactc cag 109 Met Ser Phe Tyr Ser Lys Gln Asn Ser Lys Met Asp Leu Glu LeuGln 1 5 10 15 gat ttt tat aac aag aca ctt gcc aca gag aac aat acg gctgcc act 157 Asp Phe Tyr Asn Lys Thr Leu Ala Thr Glu Asn Asn Thr Ala AlaThr 20 25 30 cgg aat tct gat ttc cca gtc tgg gat gac tat aaa agc agt gtagat 205 Arg Asn Ser Asp Phe Pro Val Trp Asp Asp Tyr Lys Ser Ser Val Asp35 40 45 gat tta cag tat ttt ctg att gga ctt tat aca ttt gta agt ctt ctc253 Asp Leu Gln Tyr Phe Leu Ile Gly Leu Tyr Thr Phe Val Ser Leu Leu 5055 60 ggt ttt atg ggg aat cta ctt att tta atg gct ctc atg aga aag cgt301 Gly Phe Met Gly Asn Leu Leu Ile Leu Met Ala Leu Met Arg Lys Arg 6570 75 80 aat cag aag acg atg gta aac ttc ctc ata gga aat ttg gcc ttc tct349 Asn Gln Lys Thr Met Val Asn Phe Leu Ile Gly Asn Leu Ala Phe Ser 8590 95 gat att ttg gtt gtg ctg ttt tgc tca cct ttt aca ctg acc tct gtc397 Asp Ile Leu Val Val Leu Phe Cys Ser Pro Phe Thr Leu Thr Ser Val 100105 110 ctg ctg gat cag tgg atg ttt ggc aaa gtc atg tgt cac att atg cct445 Leu Leu Asp Gln Trp Met Phe Gly Lys Val Met Cys His Ile Met Pro 115120 125 ttt ctt caa tgt gtg tca gtt ctg gtt tca act tta att cta ata tca493 Phe Leu Gln Cys Val Ser Val Leu Val Ser Thr Leu Ile Leu Ile Ser 130135 140 att gcc att gtc agg tat cat atg atc aag cat cct ata tct aat aat541 Ile Ala Ile Val Arg Tyr His Met Ile Lys His Pro Ile Ser Asn Asn 145150 155 160 tta aca gca aac cat ggc tac ttc ctg att gct act gtc tgg acacta 589 Leu Thr Ala Asn His Gly Tyr Phe Leu Ile Ala Thr Val Trp Thr Leu165 170 175 ggt ttt gcg att tgt tct ccc ctt cca gtg ttt cac agt ctg gtggaa 637 Gly Phe Ala Ile Cys Ser Pro Leu Pro Val Phe His Ser Leu Val Glu180 185 190 ctt cag gaa aca ttt gac tcc gca ttg ctg agc agc agg tat ttatgt 685 Leu Gln Glu Thr Phe Asp Ser Ala Leu Leu Ser Ser Arg Tyr Leu Cys195 200 205 gtt gag tcg tgg cca tct gat tcg tac aga atc gct ttt act atctct 733 Val Glu Ser Trp Pro Ser Asp Ser Tyr Arg Ile Ala Phe Thr Ile Ser210 215 220 tta ttg cta gtc cag tat att ctt ccc ttg gtg tgt cta act gtgagc 781 Leu Leu Leu Val Gln Tyr Ile Leu Pro Leu Val Cys Leu Thr Val Ser225 230 235 240 cat acc agt gtc tgc agg agt ata agc tgc ggg ttg tcc aacaaa gaa 829 His Thr Ser Val Cys Arg Ser Ile Ser Cys Gly Leu Ser Asn LysGlu 245 250 255 aac aaa ctg gaa gaa aac gag atg atc aac tta act ctt caacca ttc 877 Asn Lys Leu Glu Glu Asn Glu Met Ile Asn Leu Thr Leu Gln ProPhe 260 265 270 aaa aag agt ggg cct cag gtg aaa ctt tcc agc agc cat aaatgg agc 925 Lys Lys Ser Gly Pro Gln Val Lys Leu Ser Ser Ser His Lys TrpSer 275 280 285 tat tca ttc atc aga aaa cac agg aga agg tac agc aag aagacg gcg 973 Tyr Ser Phe Ile Arg Lys His Arg Arg Arg Tyr Ser Lys Lys ThrAla 290 295 300 tgt gtc tta cct gct cca gca aga cct cct caa gag aac cactca aga 1021 Cys Val Leu Pro Ala Pro Ala Arg Pro Pro Gln Glu Asn His SerArg 305 310 315 320 atg ctt cca gaa aac ttt ggt tct gta aga agt cag cattct tca tcc 1069 Met Leu Pro Glu Asn Phe Gly Ser Val Arg Ser Gln His SerSer Ser 325 330 335 agt aag ttc ata ccg ggg gtc ccc acc tgc ttt gag gtgaaa cct gaa 1117 Ser Lys Phe Ile Pro Gly Val Pro Thr Cys Phe Glu Val LysPro Glu 340 345 350 gaa aac tcg gat gtt cat gac atg aga gta aac cgt tctatc atg aga 1165 Glu Asn Ser Asp Val His Asp Met Arg Val Asn Arg Ser IleMet Arg 355 360 365 atc aaa aag aga tcc cga agt gtt ttc tat aga cta accata ctg ata 1213 Ile Lys Lys Arg Ser Arg Ser Val Phe Tyr Arg Leu Thr IleLeu Ile 370 375 380 cta gtg ttt gcc gtt agc tgg atg cca cta cac ctt ttccat gtg gta 1261 Leu Val Phe Ala Val Ser Trp Met Pro Leu His Leu Phe HisVal Val 385 390 395 400 act gat ttt aat gac aac ctc att tca aac agg catttc aaa ttg gtg 1309 Thr Asp Phe Asn Asp Asn Leu Ile Ser Asn Arg His PheLys Leu Val 405 410 415 tat tgc att tgt cat ttg tta ggc atg atg tcc tgttgt ctt aat cct 1357 Tyr Cys Ile Cys His Leu Leu Gly Met Met Ser Cys CysLeu Asn Pro 420 425 430 att ctg tat ggt ttt ctc aat aat ggg atc aaa gctgat tta att tcc 1405 Ile Leu Tyr Gly Phe Leu Asn Asn Gly Ile Lys Ala AspLeu Ile Ser 435 440 445 ctt ata cag tgt ctt cat atg tca taa ttcttcatgtttaccaagga 1452 Leu Ile Gln Cys Leu His Met Ser 450 455 gacaacaaatgttgggatcg tctaaaa 1479 14 456 PRT Canine 14 Met Ser Phe Tyr Ser Lys GlnAsn Ser Lys Met Asp Leu Glu Leu Gln 1 5 10 15 Asp Phe Tyr Asn Lys ThrLeu Ala Thr Glu Asn Asn Thr Ala Ala Thr 20 25 30 Arg Asn Ser Asp Phe ProVal Trp Asp Asp Tyr Lys Ser Ser Val Asp 35 40 45 Asp Leu Gln Tyr Phe LeuIle Gly Leu Tyr Thr Phe Val Ser Leu Leu 50 55 60 Gly Phe Met Gly Asn LeuLeu Ile Leu Met Ala Leu Met Arg Lys Arg 65 70 75 80 Asn Gln Lys Thr MetVal Asn Phe Leu Ile Gly Asn Leu Ala Phe Ser 85 90 95 Asp Ile Leu Val ValLeu Phe Cys Ser Pro Phe Thr Leu Thr Ser Val 100 105 110 Leu Leu Asp GlnTrp Met Phe Gly Lys Val Met Cys His Ile Met Pro 115 120 125 Phe Leu GlnCys Val Ser Val Leu Val Ser Thr Leu Ile Leu Ile Ser 130 135 140 Ile AlaIle Val Arg Tyr His Met Ile Lys His Pro Ile Ser Asn Asn 145 150 155 160Leu Thr Ala Asn His Gly Tyr Phe Leu Ile Ala Thr Val Trp Thr Leu 165 170175 Gly Phe Ala Ile Cys Ser Pro Leu Pro Val Phe His Ser Leu Val Glu 180185 190 Leu Gln Glu Thr Phe Asp Ser Ala Leu Leu Ser Ser Arg Tyr Leu Cys195 200 205 Val Glu Ser Trp Pro Ser Asp Ser Tyr Arg Ile Ala Phe Thr IleSer 210 215 220 Leu Leu Leu Val Gln Tyr Ile Leu Pro Leu Val Cys Leu ThrVal Ser 225 230 235 240 His Thr Ser Val Cys Arg Ser Ile Ser Cys Gly LeuSer Asn Lys Glu 245 250 255 Asn Lys Leu Glu Glu Asn Glu Met Ile Asn LeuThr Leu Gln Pro Phe 260 265 270 Lys Lys Ser Gly Pro Gln Val Lys Leu SerSer Ser His Lys Trp Ser 275 280 285 Tyr Ser Phe Ile Arg Lys His Arg ArgArg Tyr Ser Lys Lys Thr Ala 290 295 300 Cys Val Leu Pro Ala Pro Ala ArgPro Pro Gln Glu Asn His Ser Arg 305 310 315 320 Met Leu Pro Glu Asn PheGly Ser Val Arg Ser Gln His Ser Ser Ser 325 330 335 Ser Lys Phe Ile ProGly Val Pro Thr Cys Phe Glu Val Lys Pro Glu 340 345 350 Glu Asn Ser AspVal His Asp Met Arg Val Asn Arg Ser Ile Met Arg 355 360 365 Ile Lys LysArg Ser Arg Ser Val Phe Tyr Arg Leu Thr Ile Leu Ile 370 375 380 Leu ValPhe Ala Val Ser Trp Met Pro Leu His Leu Phe His Val Val 385 390 395 400Thr Asp Phe Asn Asp Asn Leu Ile Ser Asn Arg His Phe Lys Leu Val 405 410415 Tyr Cys Ile Cys His Leu Leu Gly Met Met Ser Cys Cys Leu Asn Pro 420425 430 Ile Leu Tyr Gly Phe Leu Asn Asn Gly Ile Lys Ala Asp Leu Ile Ser435 440 445 Leu Ile Gln Cys Leu His Met Ser 450 455 15 23 DNA ArtificialSequence Description of Artificial Sequence primer 15 gccttttcttcaatgtgtgt cag 23 16 26 DNA Artificial Sequence Description ofArtificial Sequence primer 16 ccagacagta gcaatcagga agtagc 26 17 25 DNAArtificial Sequence Description of Artificial Sequence primer 17aagcttctag agatccctcg acctc 25 18 25 DNA Artificial Sequence Descriptionof Artificial Sequence primer 18 aggcgcagaa ctggtaggta tggaa 25 19 33DNA Artificial Sequence Description of Artificial Sequence primer 19gaactctaag atggatttag aactccagat ttt 33 20 26 DNA Artificial SequenceDescription of Artificial Sequence primer 20 atgcttccgg ctcgtatgttgtgtgg 26 21 26 DNA Artificial Sequence Description of ArtificialSequence primer 21 gcctcttcgc tattacgcca gctggc 26 22 18 DNA ArtificialSequence Description of Artificial Sequence primer 22 tagtcatcccagactggg 18 23 29 DNA Artificial Sequence Description of ArtificialSequence primer 23 gtagtctccc tctcagaatt gatttatcg 29 24 32 DNAArtificial Sequence Description of Artificial Sequence primer 24ggtaaacatg aagaattatg acatatgaag ac 32

What is claimed is:
 1. A process for determining whether a chemicalcompound specifically binds to and activates a Y5 receptor, whichcomprises contacting nonneuronal cells transfected with and expressingDNA encoding a human, rat, or canine Y5 receptor with the chemicalcompound under conditions suitable for activation of the Y5 receptor,and measuring the binding of GTPγS to the cells in the presence and inthe absence of the chemical compound, a change in the binding of GTPγSin the presence of the chemical compound indicating that the chemicalcompound activates the Y5 receptor, wherein such cells prior to beingtransfected with such DNA do not express the human, rat, or canine Y5receptor, and wherein the human Y5 receptor has an amino acid sequenceidentical to the amino acid sequence shown in FIG. 6 (SEQ ID NO: 4) orthat encoded by plasmid pcEXV-hY5 (ATCC Accession No. 75943); the rat Y5receptor has an amino acid sequence identical to the amino acid sequenceshown in FIG. 4 (SEQ ID NO: 2) or that encoded by plasmid pcEXV rY5(ATCC Accession No. 75944); and the canine Y5 receptor has an amino acidsequence identical to the amino acid sequence shown in FIG. 24 (SEQ IDNO: 14) of that encoded by plasmid cY5-B011 (ATCC Accession No. 97587).2. A process for determining whether a chemical compound specificallybinds to and activates a Y5 receptor which comprises contacting amembrane fraction from nonneuronal cells transfected with and expressingDNA encoding a human, rat, or canine Y5 receptor, with the chemicalcompound under conditions suitable for activation of the Y5 receptor,and measuring the binding of GTPγS to the membrane fraction in thepresence and in the absence of the chemical compound, a change in thebinding of GTPγS in the presence of the chemical compound indicationthat the chemical compound activates the Y5 receptor, wherein such cellsprior to being transfected with such DNA do not express the human, rat,or canine Y5 receptor, and wherein the human Y5 receptor has an aminoacid sequence identical to the amino acid sequence shown in FIG. 6 (SEQID NO: 4) or that encoded by plasmid pcEXV-hY5 (ATCC Accession NO.75943), the rat Y5 receptor has an amino acid sequence identical to theamino acid sequence shown in FIG. 4 (SEQ ID NO: 2) or that encoded byplasmid pcEXV-rY5 (ATCC Accession No. 75944); and the canine Y5 receptorhas an amino acid sequence identical to the amino acid sequence shown inFIG. 24 (SEQ ID NO: 14) or that encoded by plasmid cY5-B011 (ATCCAccession No. 97587).
 3. A process for determining whether a chemicalcompound specifically binds to and inhibits activation of a Y5 receptor,which comprises contacting nonneuronal cells transfected with andexpressing DNA encoding a human, rat, or canine Y5 receptor, with boththe chemical compound and a second chemical compound known to activatethe Y5 receptor, and separately with only the second chemical compound,under conditions suitable for activation of the Y5 receptor, andmeasuring binding of GTPγS to the cells in the presence of only thesecond chemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in GTPγS binding inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compoundindicating that the chemical compound inhibits activation of a Y5receptor, wherein such cells prior to being transfected with such DNA donot express the human, rat, or canine Y5 receptor, and wherein the humanY5 receptor has an amino acid sequence identical to the amino acidsequence shown in FIG. 6 (SEQ ID NO: 4) or that encoded by plasmidpcEXV-hY5 (ATCC Accession No. 75943); the rat Y5 receptor has an aminoacid sequence identical to the amino acid sequence shown in FIG. 4 (SEQID NO: 2) or that encoded by plasmid pcEXV-rY5 (ATCC Accession No.75944); and the canine Y5 receptor has an amino acid sequence identicalto the amino acid sequence shown in FIG. 24 (SEQ ID NO: 14) or thatencoded by plasmid cY5-B011 (ATCC Accession No. 97587).
 4. A process fordetermining whether a chemical compound specifically binds to andinhibits activation of a Y5 receptor, which comprises contacting amembrane fraction from a cell extract of nonneuronal cells transfectedwith and expressing DNA encoding a human, rat, or canine Y5 receptor,with both the chemical compound and s second chemical compound known toactivate the Y5 receptor, and separately with only the second chemicalcompound, and measuring binding of the GTPγS to the membrane fraction inthe presence of only the second chemical compound and in the presence ofboth the chemical compound and the second chemical compound, a smallerchange in GTPγS binding in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of a Y5 receptor, wherein such cells prior to beingtranstected with such SNA do not express the human, rat, or caninge Y5receptor, and wherein the human Y5 receptor has an amino acid sequenceidentical to the amino acid sequence shown in FIG. 6 (SEQ ID NO: 4) orthat encoded by plasmid pcEXV-hY5 (ATCC Accession No. 75943); the rat Y5receptor has an amino acid sequence identical to the amino acid sequenceshown in FIG. 4 (SEQ ID NO: 2) or that encoded by plasmid pcEXV-rY5(ATCC Accession No. 75944); and the canine Y5 receptor has an amino acidsequence identical to the amino acid sequence shown in FIG. 24 (SEQ IDNO: 14) or that encoded by plasmid cY5-B011 (ATCC Accession No. 97587).5. The process of claims 1 or 2, wherein the change is an increase inGTPγS binding.
 6. A process of any of claims 1, 2, 3 or 4, wherein thecells are insect cells.
 7. A process of any of claims 1, 2, 3 or 4,wherein the cells are mammalian cells.
 8. The process of claim 7,wherein the mammalian cells are COS-7 cells, CHO cells, 293 humanembryonic kidney cells, NIH-3T3 cells or LM(tk-) cells.
 9. A method ofpreparing a pharmaceutical composition which comprises determiningwhether a compound (i) binds to, and (ii) activates or inhibitsactivation of, a Y5 receptor using the process of any of claims 1, 2, 3,or 4, recovering the compound free of any Y5 receptor, and admixing thecompound with a pharmaceutically acceptable carrier.